Non-Imuunosuppressive cyclosporins and their use in the prevention and treatment of HIV infection

ABSTRACT

Disclosed are cyclosporin analogs having amino acid residue substitutions at positions 1, 3, or 7 of the cyclosporin peptide backbone. Also disclosed are conjugates of these cyclosporin analogs in which an HIV protease inhibitor moiety is conjugated to the position-7 amino acid residue of the cyclosporin. These compounds simultaneously bind to and inhibit cyclophilin and HIV protease. The compounds have good bioavailability and potent HIV inhibitory activity. They are useful in the treatment and prevention of HIV-mediated disorders, including AIDS.

Priority is claimed to provisional application Ser. No. 60/057,751,filed Aug. 26, 1997.

GOVERNMENT RIGHTS

This invention was made with United States government support awarded bythe following agencies: NIH Grant Nos: AR32007 and GM50113. The UnitedStates has certain rights in this invention.

FIELD OF THE INVENTION

The invention is directed to non-immunosuppressive cyclosporin analogsand conjugates thereof which possess anti-HIV activity. The compoundsand compositions containing the same are useful in the prevention andtreatment of HIV infection in humans.

DESCRIPTION OF THE PRIOR ART

Cyclosporin A (CsA) 1.1, marketed by Sandoz under the trademark“SANDIMMUNE,” currently is the drug of choice for preventing rejectionof transplanted human organs. CsA is a highly lipophilic, cyclicundecapeptide,cyclo(-MeBmt¹-Abu²-Sar³-MeLeu⁴-Val⁵-MeLeu⁶-Ala⁷-(D)-Ala⁸-MeLeu⁹-MeLeu¹⁰-MeVal¹¹-)a(SEQ. ID. NO: 1), that contains 7 N-methyl amino acid residues and thenovel amino acid (4R)-4-{(E)-2-butenyl}-4-N-methyl-(L)-threonine(abbreviated as MeBmt) in the 1-position. A number of synthetic routesare known in the art for solution-phase or solid-phase synthesis of CsA.See, for example, Rich et al. (1995), “Solid Phase Synthesis ofCyclosporin Peptides.” J. Am. Chem. Soc. 117:7279-7280; Wenger, R. M.(1984), Helv. Chim. Acta 67:502; and Wenger, R. M. (1985), Angew. Chem.Int. Ed. Engl. 24:77. CsA is depicted in structure 1.1:

CsA is produced by the fungus Tolypocladium niveum and was firstisolated in 1976 by workers at Sandoz. In 1983, CsA was approved by theU.S. Food and Drug Administration for use as an immunosuppressant in theUnited States.

The structure of CsA has been confirmed by total synthesis, Wenger(1984), Helv. Chim. Acta, 67:502, and the conformations of CsA free insolution and bound to the protein cyclophilin have been solved by NMRand X-ray crystallography. Looslie et al. (1985), Helv. Chim. Acta,68:682 and Mikol (1993), J. Mol. Biol., 234:1119, respectively.

Several modified cyclosporin derivatives are described in the prior art.A shorthand notation for designating cyclosporin analogs has developedin which any modified amino acids and their positions relative tounmodified CsA are listed. This makes for a very simple and unambiguousdesignation of cyclosporin analogs based upon their differences fromnatural CsA. For example, an analog of CsA possessing a serine residuein place of the normal valine as the fifth amino acid residue isdesignated (Ser⁵)-CsA. This conventional shall be consistently employedherein.

CsA analogs containing modified amino acids in the 1-position arereported by Rich et al. (1986), J. Med. Chem., 29:978. Stronglyimmunosuppressive, anti-inflammatory, and anti-parasitic CsA analogs aredescribed in U.S. Pat. Nos. 4,384,996; 4,771,122; and 5,284,826, allassigned to Sandoz. Among the CsA analogs described in these patents are(AllylGly²)-CsA, ((D)-Ser⁸)-CsA, and (O-(2-hydroxyethyl)(D)Ser⁸) CsA.

In 1984, Handschumacher et al. reported the discovery of a CsA bindingprotein, named cyclophilin (Cyp), that binds CsA with a dissociationconstant of approxinately 20 nM. Handschumacher et al. (1984) Science226:544. It was later shown that Cyp is homologous with peptidylprolylisomerase (PPIase) a ubiquitous family of proteins found in avariety of cell types. See Takahashi (1989), Nature 337:473 and Fischeret al. (1989) Nature 337:476. Cyclophilins catalyze the cis-transisomerization of Xaa-Pro bonds and are hypothesized to play a role inprotein folding, although this functionality remains uncertain. See, forinstance, Fischer (1994), Angew. Chem. Int. Ed. Engl. 33:1415 and Schmid(1993), Ann. Rev. Biophys. Biomol. Struct. 22:123.

The identification of Cyp as a PPIase suggested that CsA exerts itsimmunosuppressive effect by inhibiting the PPIase activity of Cyp,thereby causing improper folding of proteins which are crucial to theimmune response. Signal et al. (1991), J. Exp. Med. 173:619. Thishypothesis was originally strengthened by the discovery that themacrolide FK506, 1.2, has potent immunosuppressive activity and inhibitsthe PPIase activity of FK506 binding protein (FKBP). Siekerka et al.(1989), Nature 341:755.

Further investigations, however, revealed several discrepanciesregarding the inhibition of PPIase as a mechanism leading toimmunosuppression. Foremost, the concentrations of CsA and FK506required to ellicit immunosuppression are far lower than theconcentrations of Cyp within a cell. Additionally, mutants of yeast andneurospora which lack the Cyp gene are resistant to cyclosporin but arestill viable. See Agarwal et al. (1987), Transplantation 42:627;Tropschung et al. (1989), Nature 342:953; and Hayano et al. (1991),Biochem. 30:3041. Another observation at odds with the originalhypothesis was that although CsA and FK506 exhibit very similar in vivoand in vitro effects, CsA does not bind to FKBP and FK506 does not bindto Cyp. Schreiber and Crabtree (1992), Immunology Today 13:136. ThePPIase inhibition hypothesis was further weakened with the discoverythat several potent PPIase inhibitors do not cause immunosuppression.See, for example, Somers et al. (1991), J. Am. Chem. Soc. 113:8045.

In 1991, Liu et al. reported that the CsA-Cyp complex binds with highaffinity to calcineurin, a calcium dependent serine/threoninephosphatase, Liu et al. (1991), Cell 66:807; and Liu et al. (1992),Biochem. 31:3896. Calcineurin is thought to cleave a phosphate groupfrom the nuclear factor of activated T-cells (NF-AT), allowing itstranslocation into the nucleus where it activates the gene forinterleukin-2. See Schreiber and Crabtree (1992), supra. Inhibition ofcalcineurin is now generally accepted as the mechanism ofimmunosuppression by both CsA and FK506. See, for example, Ho et al.(1996), Clin. Immun. and Immunopathology, 80:S40. CsA binds to Cyp by aninteraction between residues 9-10-11-1-2 of the CsA and an active siteon Cyp residues. 9-10-11-1-2 of CsA are therefore referred to as the“binding domain.” Calcineurin is bound to CsA by an analogousinteraction including residues 4-5-6-7-8 of CsA. These residues aretherefore referred to as the “effective domain”:

In the late 1980's, CsA was reported to exhibit anti-HIV activity. See,for instance, Wainberg et al. (1988), Blood, 72:1904; Karpas et al.(1992), Proc. Natl. Acad. Sci. USA, 89:8351; and Bell et al. (1993),Proc. Natl. Acad. Sci. USA, 90:1411. Although it originially seemedcounterintuitive to use an immunosuppressant suppressant to treat aviral infection that compromises the immune system, the anti-HIVactivity of CsA was at first attributed to the inhibition of T-cellactivation. However this hypotheses was disproved whennon-immunosuppressive CsA analogs were also found to have anti-HIVactivity. Bartz et al. (1995), Proc. Natl. Acad. Sci. USA, 92:5381; andRosenwirth et al. (1994), Antimicrobial Agents and Chemotherapy,38:1763.

Regarding HIV and its replication in human T-cells, HIV protease is anaspartic protease that cleaves the immature viral protein gag-pol intomature structural proteins and enzymes. HIV protease is an essentialenzyme in the replication of the HIV virus. Katz et al. (1994), AnnualRev. Biochem., 63:133. A decade of intense research has produced fourFDA-approved HIV protease inhibitors, saquinivar 1.9, ritonavir 1.10,indinivar 1.11, and nelfinivar 1.12; and one compound currently in phaseIII trials, VX-478 1.13. The structure of these compounds are shownbelow:

Unfortunately, initial reports of clinical success by treatment with asingle protease inhibitor have been tempered by the rapid onset ofresistance. Molla et al. (1996), Nature Medicine, 2:760. Due to the poorfidelity of reverse transcriptase, the enzyme that producesdouble-stranded DNA from the viral RNA, a large number of geneticmutations of the virus are produced. The low fidelity of the reversetranscriptase reaction is compounded by the massive turnover of viralparticles during the HIV life cycle. Ho et al. (1996), Science,271:1582, have calculated that the decay half-life of virions is on theorder of 0.24 days and that for infected cells the decay half-life is1.5 days. These numbers indicate that every six hours approximatelyone-half of the circulating virus is removed and replenished and thatapproximately 10.3 billion virions are produced and released into thebloodstream each day in an infected individual. Due to the low fidelityof the reverse transcriptase, mutations will occur at virtually everyposition in the viral genome, along with some double mutants. The rapidemergence of resistance to single-compound therapies is therefore notsurprising.

Although the first generation protease inhibitors, in combination withreverse transcriptase inhibitors, have provided the most effectiveanti-HIV therapies to date, there is still a need for more potent HIVprotease inhibitors which have increased bioavailability and half-life,and less susceptibility to loss of efficacy due to mutations in thevirus.

Recently, DuPont-Merck researchers reported several cyclic ureas thatbind tightly to HIV protease in nanomolar concentrations. However,clinical trials on one of these compounds (DMP-323) 1.14 werediscontinued to poor water solubility and first-pass metabolism. DeLucca(1997), Drug Disc. Today, 2:6). DMP-450 1.15, a second generation cyclicurea, has comparable potency to DMP-323 and achieves much higher plasmaconcentrations. Another cyclic urea, designated SD-146 1.16 displaysexcellent anti-HIV potency (IC₉₀=5.1 nM). However, the compound isextremely insoluble in both water and most oils. Jadhav et al. (1997),J. Med. Chem., 40:181. The structure of these cyclic urea HIV proteaseinhibitors are shown below:

Other non-peptide HIV protease inhibitors have been reported byinvestigators at Upjohn and Gilead:

SUMMARY OF THE INVENTION

In a first embodiment, the invention is directed to non-immunosupressivecyclosporins comprising a cyclic undecapeptide of Formula I:

wherein V is a MeLeu(3-OH), MeLeu, MeSer, MeSer-PG, MeThr, or MeThr-PGresidue; W is a (D)-N-methyl-amino acid residue (or N-methylglycyl,which is non-chiral), preferably (D)-N-methylserinyl, or(D)-N-methylserinyl-PG, wherein each PG is, independently, a side-chainprotecting group; X and X′ are independently an N-methyl-leucinyl or anN-methylalanyl residue; Y is a lysyl, homo-lysyl, ornithinyl, lysyl-PG,homo-lysyl-PG, or ornithinyl-PG residue, wherein each PG is,independently, a side-chain protecting group; and Z is absent or is anHIV protease inhibitor moiety conjugated to Y via a side-chain on Y; andsalts thereof.

In the preferred embodiments, V is a MeLeu(3-OH) residue, W is a(D)-N-methylserinyl residue, X and X′ are N-methyl-leucinyl residues, Yis a lysyl residue, and Z is selected from the group consisting of:

wherein R is Ac-Ser-Leu-Asn or Cbz-Asn.

In a second embodiment, the invention is also drawn to pharmaceuticalcompositions for the prevention and treatment of HIV-mediated disorders,including AIDS, in humans. The pharmaceutical compositions contain aneffective HIV protease-inhibiting amount of one or more of the Formula Icompounds or pharmaceutically-acceptable salts of the compounds. Thepreferred pharmaceutical composition contains the Formula I compoundwherein W is a (D)-N-methylserinyl residue, X and X′ areN-methyl-leucinyl residues, Y is a lysyl residue and Z is

wherein R is Cbz-Asn.

A third embodiment of the invention is drawn to a method of treatingHIV-mediated disorders, including AIDS, in humans. The method comprisesadministering to a human subject in need thereof an effective HIVprotease-inhibiting amount of one or more compounds of Formula I, orpharmaceutically-acceptable salts thereof.

A fourth embodiment of the invention is directed to a compoundcomprising a conjugate which is produced by forming a cyclosporin analogcomprising a lysyl residue having an ε-aminobutyl side-chain at7-position of the cyclosporin analog; and then conjugating an HIVprotease inhibitor to the ε-aminobutyl side-chain of the lysyl residue.These conjugates simultaneously bind to and inhibit the action ofcyclophilin and HIV protease. Consequently, they are useful in thetreatment of HIV-mediated disorders, including AIDS.

A distinct advantage of the present invention is that it provides novelCsA analogs which are non-immunosuppresive and which are potentinhibitors of HIV protease. These compounds are therefore highlyeffective inhibitors of HIV replication.

Another advantage of the invention is that it provides a generalizedapproach for preparing compounds which are effective to inhibit HIVprotease by conjugating an HIV protease inhibitor moiety to a CsA analoghaving a lysyl residue in the 7-position of the cyclosporin skeleton.When conjugated to this position, the inhibitor moiety does notinterfere with the ability of the CsA to bind to and inhibit the PPIaseactivity of the Cyp, while simultaneously enabling the conjugate to bindto and inhibit HIV protease. Because the ε-aminobutyl side-chain of the7-position lysyl residue extends away from the CsA ring, virtually anymolecule can be attached to the aminobutyl side-chain without adverselyaffecting the binding of the CsA analog to Cyp.

A notable benefit to this approach is that compounds which exhibit HIVprotease inhibitor activity, when conjugated to the CsA analog, displayincreased bioavailability and cell penetration and are targeted to Cyp,an enzyme believed to play an as yet undetermined role in thereplication of the HIV. This places the inhibitor moiety in a positionto disrupt the HIV replication cycle by inhibiting HIV protease, whilethe “binding domain” of the CsA analog simultaneously binds to andinhibits Cyp.

A striking aspect of the invention which is confirmed by the NationalCancer Institute screenings presented in the Examples is that thesubject analogs and conjugates are taken into cells. This is remarkablebecause the molecular weight of the conjugates is on the order of 2,000Da. It is commonly accepted that compounds having a molecular weightgreater than about 500 Da are only poorly taken into cells.Consequently, a primary utility of the invention is to take compoundshaving a good anti-HIV activity, but poor bioavailability, andconjugating them to a CsA analog, thereby increasing the bioavailabilityand anti-HIV activity of the compound. The invention can be practicedwith any anti-HIV moiety which can be conjugated to an amino moiety.

Another advantage to the compounds of the invention is that by insertinga MeLeu(3-OH) residue in the 1-position, the compounds are renderednon-immunosuppressive but maintain their anti-HIV activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the anti-HIV activity of the compound 2.8according to the U.S. National Cancer Institute's “In Vitro Anti-AIDSDrug Discovery Program.”

FIG. 2 is a graph depicting the anti-HIV activity of compound 2.6 asdetermined by the same assay as in FIG. 1.

FIG. 3 is a graph depicting the anti-HIV activity of compound 2.10 asdetermined by the same assay as in FIG. 1.

FIG. 4 is a graph depicting the anti-HIV activity of compound 4.20 asdetermined by the same assay as in FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions

To aid in a consistent understanding of the invention, the followingabbreviations and definitions shall be used herein:

AIDS: Acquired Immune Deficiency Syndrome, as defined by the UnitedStates Centers for Disease Control.

Binding Domain of CsA: The sub-domain of cyclosporin and cyclosporinanalogs which binds to cyclophilin. The binding domain generallyincludes residues 9-10-11-1-2 of cyclosporin.

Cyclophilin (Cyp): A ubiquitous cytosolic protein having PPIaseactivity. Cyclosporins bind to and inhibit the action of cyclophilin.

Cyclosporin A (CsA): As used herein, the terms “cyclosporin,”“cyclosporin derivative,” and “cyclosporin analog” denote any compoundhaving the fundamental structure of cyclosporin A, namely a cyclicundecapeptide having a number of N-methyl-substituted amino acidresidues, with amino acid substitutions at defined positions within themolecule.

Cyclosporin Conjugates: A CsA analog or derivative conjugated to anexocyclic moiety, especially but not exclusively at position-7 of theCsA skeleton. Cyclosporin conjugates are molecules which retain theability to bind to cyclophilin.

Effector Domain of CsA: In unmodified CsA, the sub-domain which binds tocalcineurin. Generally includes residues 4-5-6-7-8 of the CsA molecule.In CsA analogs and conjugates, the effector domain is the region of theCsA skeleton where various anti-HIV substituents are attached to the CsAmolecule to alter the specificity of the CsA analog or conjugate.

Human Immuno-Deficiency Virus (HIV): Any of a class of virus which arebelieved to be causative of AIDS in man.

HIV protease: HIV protease is an aspartic protease that cleaves theimmature viral protein gag-pol into mature structural proteins andenzymes. HIV protease is an essential enzyme in the replication of theHIV virus.

HIV protease inhibitor: Any compound now known or developed in thefuture which inhibits or prevents the action of HIV protease, therebyinhibiting or preventing the replication of HIV.

Modified Residues, Reagents, Protecting Groups, and Solvents:Abu=α-aminobutyric acid, Bn=benzyl, Boc=t-butoxycarbonyl,BOP=benzotriazolyl-N-oxy-tris(dimethylamino)phosphoniumhexaflurophosphate BOP-Cl=bis(2-oxo-3-oxazolidinyl phosphonic chloride,Cbn=benzylcarbonyl, Cbz=benzyloxycarbonyl, DCC=dicyclohexylcarbodiimide, DCM=dichloromethane, DCU=dicyclohexylurea,DIBAL=diisobutylaluminum hydride, DIEA=diisopropylethylamine,DIPCDI=diisopropylcarbodiimide, DMAP=4-dimethylaminopyridine,DMF=dimethylformamide, EthOAc=ethylacetate, FABMS=Fast Atom BombardmentMass Spectrometry, Fmoc=9-fluorenylmethoxycarbonyl,HATU=O-(7-azabenzotriazol-1-yl)-1,1,2,2-tetramethyluroniumhexafluorophosphate, HEA=hydroxyethyl amine,HOBt=1-hydroxybenzotriazole, LAH=lithium alumunium hydroxide,MeLeu(3-OH)=3-hydroxy-N-methyl-leucine, MTBE=methyl-t-butyl ether, NMM:N-methylmorpholine, NMP=N-methyl pyrrolidinone, OBn=benzyloxy,OTBS=t-butyldimethylsilyloxy, PyAOP=7-azabenzotriazole-1-yl oxytris(pyrodidino) phosphonium hexafluorophosphate, Sar=sarcosine,TBAF=tetrabutylammonium fluoride, TBS=t-butyldimethylsilyl,TBS-Cl=t-butyldimethylsilyl chloride, TEA=triethyl amine,TFA=trifluoroacetic acid, THF=tetrahydrofuran, TMS=tetramethylsilyl,TLC=thin layer chromatography, Trt=trityl (i.e., triphenylmethyl).

Peptidyl prolylisomerase (PPIase): The functional class of enzymes towhich cyclophilin belongs. PPIase catalyzes the cis-trans conversion ofXaa-Pro bonds.

Overview

The invention is drawn to CsA analogs and conjugates whichsimultaneously bind to cyclophilin and HIV protease in a biologicalsystem. In particular, the invention encompasses a class of CsA analogsin which amino acid substitutions have been made at the 1, 3, and7-positions of the CsA skeleton. By judiciously altering the structureof natural CsA, the CsA analogs described herein arenon-immunosuppresive and strongly inhibitory of HIV replication as shownin a standard, widely-accepted assay (see the Examples).

The invention is also drawn to conjugates of these CsA analogs. In itsmost general approach, the invention is drawn to conjugating an HIVprotease inhibitor to a residue within the effector domain of the CsAskeleton, preferably the residue at position-7. This allows the HIVprotease moiety to be exposed to solution, even when the binding domainof the CsA molecule is joined to Cyp. Because Cyp is believed to play arole in HIV replication, conjugating an HIV protease inhibitor moiety tothe effector domain of CsA disposes the inhibitor in close proximity tothe HIV virion during replication (or likewise disposes thenon-immunosuppresive CsA analog in close proximity to the HIV virionduring replication), thereby increasing its ability to inhibit thereplication process.

Additionally, because endogenous Cyp levels are much higher than HIVprotease levels in infected cells, higher levels of the conjugated HIVprotease inhibitor are attainable than are using the isolated proteaseinhibitor itself. This further aids in inhibiting HIV replication.

While not being limited to any particular molecular mechanism, it isbelieved that the conjugates assert their anti-HIV action bysimultaneously binding to and inhibiting both Cyp and HIV protease.

What follows is a description of the chemical reactions by which the CsAanalogs and conjugates are made, followed by a description ofincorporating the analogs and conjugates into pharmaceuticalcompositions for the treatment of HIV-mediated disorders. The “Examples”section includes illustrative synthetic protocols and thirdparty-generated data showing the anti-HIV activity of the compounds.

Chemistry

Synthesis of (2S,3R)-MeLeu(3-OH)

Route 1: The first route to (2S,3R)-MeLeu(3-OH), is based on a(2R,3R)-Leu(3-OH) synthesis reported by Evans et al (1987), TetrahedronLett., 28:39 uses a boron enolate mediated Evans aldol reaction and anefficient epimerization of a cis- to trans-oxazolidonone as the keysteps (Scheme 3.3). Boron triflate-mediated condensation of thebromoacetyl chiral auxiliary 3.7, obtained from the chiral auxiliary in82% yield, with isobutrylaldehyde yielded the aldol product 3.8 in 50%yield.

Reaction of the methylisocyanate with 3.8 catalyzed by BF₃OEt₂, producesthe carbamate 3.9 in 80% yield. Removal of the chiral auxiliary withLiOH and cyclization of the carbamate with tert-butoxide gives thecis-oxazolidinone 3.10 in 74% yield (Scheme 3.4). Reaction of acid 3.10with TMS-diazomethane gives the cis-oxazolidinone methyl ester 3.11,which is efficiently epimerized to the desired trans-oxazolidinone acid3.12 with KOH/ethanol and then hydrolysed to (2S,3R)-MeLeu(3-OH) 3.6with KOH(aq) in 73% overall yield. Although this chiral synthesis of(2S,3R)-MeLeu(3-OH) requires fewer purification steps and affords easyisolation of intermediates, the mass of the chiral auxiliary madescaling up impractical on >0.5 g reactions.

Route 2

Synthesis of (2S,3R)-MeLeu(3-OH)

A second synthesis of (2S,3R)-MeLeu(3-OH) was developed based on aLeu(3-OH) synthesis (all 4 isomers) reported by Omura et al. (1993),Tetrahedron Lett. 34:4447; and (1996), J. Am. Chem. Soc. 118:3584. TheSharpless asymmetric epoxidation reaction is used to set thestereocenters and the cis to trans-oxazolidinone epimerization describedin Route 1 is used to produce the correct stereochemistry of the finalproduct (Scheme 3.5). The α,β-unsaturated ester 3.13 is reduced withDIBAL to the allylic alcohol 3.14, which is epoxidized using D-diethyltartarate to yield the epoxy alcohol 3.15 in good yield. Treatment of3.15 with NaH and MeNCO at reflux produces a mixture of oxazolidinoneisomers, some decomposition products, and only a low yield of thedesired oxazolidinone 3.16. Oxidation of the alcohol 3.16 with a Jonesreagent gives acid 3.10, which is treated with TMS-diazomethane to givethe methyl ester 3.11 in 70% overall yield (Scheme 3.6). Epimerizationand hydrolysis of the (2R,3R) methyl ester 3.11 with KOH/ethanol gives(2S,3R) oxazolidinone acid 3.12, which is not isolated, but hydrolyseddirectly with KOH(aq) to the desired (2S,3R)-MeLeu(3-OH) 3.6.

Optimized Route to Oxazolidinone 3.16.

The synthesis of (2S,3R)-MeLeu(3-OH) in Scheme 3.5 is not optimal inthat the conversion of the epoxy alcohol 3.15 to the desiredoxazolidinone isomer 3.16 proceeds in consistently low yields. This isovercome by pre-forming the carbamate before oxazolidinone formation, byusing a stronger base in the oxazolidinone isomerization step, and byvarying the reaction temperature (Scheme 3.7). Treatment of the epoxyalcohol 3.15 with Et₃N and MeNCO produces the epoxy carbamate 3.17 inexcellent yield. Reaction of the carbamate 3.17 with NaH/THF at refluxyields the desired oxazolidinone 3.16, in the same yield as the one-potreaction. KH, a stronger base than NaH, causes significantly largeramounts of decomposition at reflux temperatures. It was found thattreatment of the epoxycarbamate 3.17 with 1.5 equivalents of KH at 0° C.for 1 hour, followed by warming to room temperature for 2 hours gives an85% yield of the products with a 4:1 ratio of the desired oxazolidinone3.16 isomer to the undesired oxazolidinone 3.18. Thus, a novel synthesisof (2S,3R)-MeLeu(3-OH) has been achieved by use of the Sharplessassymmetric epoxidation, followed by oxazolidinone isomerization withKH, and oxazolidinone epimerization to the desired trans-oxazolidinone.This synthesis is rapid, requires few purification steps, andfacilitates the large scale synthesis of (2S,3R)-MeLeu(3OH) forincorporation into CsA analogs.

Synthesis of CsA Analogs

The synthesis of the CsA analogs follows the modified Wenger procedurereported by Colucci et al. (1990), J. Org. Chem. 55:2895 (Scheme 4.1).Notable parts of the synthesis include the undecapeptide cyclizationbetween the Ala⁷-DAla⁸ residues, the “7+4” (1-7 residues+8-11 residues)segment coupling to produce the undecapeptide, and use of BOP-Cl reagentfor peptide couplings. (See “Wenger's CsA Strategy.”)

Cyclization between the alanines encounters the least steric bulk, noN-methylated residues, and an intramolecular hydrogen bonding patternthat may facilitate cyclization. The improved cyclization procedureinvolves simultaneous double deprotection of the Fmoc-protectedN-terminus and the benzyl ester-protected C-terminus of theundecapeptide with NaOH(aq)/ethanol. The undecapeptide is formed via a“7+4” segment coupling so that the valuable β-OH amino acid(MeBmt¹-(2S,3R) or MeLeu(3-OH)¹) can be incorporated late in thesynthesis. Undecapeptide formation also employs the BOP reagent whichresults in little epimerization of MeVal¹¹.

Wenger's synthesis of the “4” (8-11 position residues) segmenttetrapeptide is based on an unconventional synthetic strategy in thatthe synthesis proceeds from the left to the right in order to avoidrapid diketopiperazine formation of the H-MecLeu-MeVal-OBn dipepetideScheme 4.2. Tung et al. (1986), J. Org. Chem. 51:3350 found that byusing Fmoc/tBu protection, diketopiperazine formation is inhibited sothat conventional right to left synthesis proceeds without epimerizationof each stereogenic center (see scheme “Strategies for synthesis of 8-11CsA tetrapeptide”). Another major improvement over the Wenger procedureis the use of BOP-Cl for coupling of N-methyl amino acids. In hisoriginal synthesis of the 8-11 and 2-7 segments, Wenger used the mixedanhydride method, which requires low temperatures and long reactiontimes. The use of BOP-Cl as a N-alkyl peptide coupling reagentsignificantly improves the ease of CsA synthesis due to its manageabletemperatures (0° C. to RT) and shorter reaction times. Thus, thecombination of Wenger's overall strategy with the improved syntheticprocedures described herein produces a very powerful method forgenerating diverse CsA analogs.

Strategies for the synthesis of 8-11 CsA tetrapeptide.

Synthesis of ([MeLeu(3-OH)¹, D-MeSer (OBn)³, Lys(2Cl-Cbz)⁷])CsA

The synthesis of 1, 3, and 7-position substituted CsA analogs followsthe procedure reported by Colucci et al (Scheme 4.1), modified so thatthe 2-7 segment is synthesized linearly (Scheme 4.1) and not in a “4+2”fashion due to problems with epimerization. When the 2-3 dipeptide withD-MeSer(OBn) in the 3-position is coupled to the CsA 4-7 tetrapeptide,significant epimerization if the D-MeSer)OBn) residue was observed(Scheme 4.2). Wenger attempted to synthesize the 2-7 CsA sequencelinearly, but was unsuccessful due to rapid dikepiperazine formation ofthe Sar³-MeLeu⁴ fragment. However, we were able to synthesize theD-MeSer(OBn)³2-7 CsA peptide linearly without any observeddiketopiperazine formation. Furthermore, Seebach et al. (1993), Helv.Chim. Acta. 76:1564, has also reported a successful linear synthesis ofa 2-7 CsA analog with MeAla in the 3-position.

Lys(εN-2Cl-Cbz)⁷ and D-MeSer(OBn)³ side-chain protection was chosenbecause both protecting groups survive the acidic conditions requiredfor the CsA analog synthesis, and because hydrogenation cleaves bothgroups without decomposing the CsA analog. Furthermore, hydrogenation ofthe Lys(εN-2Cl-Cbz)⁷ produces a free amine which is used as a “handle”to attach other compounds to the CsA analog.

Synthesis of 2-7 Peptide:

Boc-MeLeu-OH was coupled with H-Lys(2Cl-Z)-OBn to yield dipeptide 4.1 ongood yield (Scheme 4.1). Cleavage of the Boc group from 4.1 withHCl/dioxane followed by reaction with Boc-Val-OH gives the tripeptide4.2 in fair yield consistent with yields reported by Colucci et al forcoupling to Boc-Val-OH. Deprotection of 4.2 and coupling withBoc-MeLeu-OH produces the 4-7 tetrapeptide 4.3 in 69% yield. Treatmentof tetrapeptide 4.3 with TFA to cleave the Boc group, neutralization ofthe salt with NaHCO_(3,) followed by coupling to Boc-D-MeSer(OBn)-OHproduces 4.4 in 71% yield. Cleavage of the Boc group from 4.4,neutralization and coupling to Boc-Abu-OH with BOP-Cl produces thehexapeptide 4.5 in 69% yield. No detectable epimerization ofdiketopiperazine formation is observed for this step. When a “4+2”coupling betweenBoc-Abu-D-MeSer(OBn)-OH+H-Meleu-Val-MeLeu-Lys(2Cl-Z)-OBn is attempted,significant epimerization of the D-MeSer(OBn) residue occurrs asexpected for an acyl-peptide coupling (Scheme 4.2).

Completion of CsA Analog

The final stages of the CsA analog synthesis follows the procedure firstdeveloped by Wenger, supra. The 2-7 hexapeptide 4.5 is coupled to theacetonide-protected MeLeu(3-OH) 4.6 with EDCI/HOBt to yield the 1-7heptapeptide 4.7 in yields of 51-70% (Scheme 4.3). Cleavage of theacetonide with standard HCl(aq) conditions and neutralization of the HClsalt with NaHCO₃ gives 4.8, which is coupled to the 8-11 tetrapeptidewith the BOP reagent to produce the undecapeptide 4.9 in 33-53% yield.Simultaneous cleavage of the Fmoc and benzyl ester groups withNaOH(aq)/ethanol followed by cyclization of the linear undecapeptidewith DMAP/Propyl phosphonic anydride in CH₂Cl₂ gives (MeLeu(3-OH)¹,D-MeSer(OBn)³, Lys(2Cl-Cbz)⁷)CsA 4.10 in fair to good yields of 30-60%(Scheme 4.4).

Synthesis of (MeLeu(3-OH)¹, D-MeSer (OH)³, Lys(2Cl-Cbz)⁷)CsA

The Boc group is cleaved from tetrapeptide 4.3, and the resulting freeamine HCl salt is coupled to Boc-D-MeSer-OH 4.12 with BOP-Cl, to givethe desired pentapeptide 4.14 (Scheme 4.6). Other coupling reagents,such as HATU or DIPCDI/HOAT can also be used (Scheme 4.6). Temporaryhydroxyl protection, which is cleaved after pentapeptide formation,improves the yield of product and the ease of purification. To this end,Boc-D-MeSer(OTBS-OH 4.13 is smoothly coupled to the tetrapeptide 4.3with BOP-Cl to give pentapeptide 4.15 in 65% yield (Scheme 4.6). The TBSgroup was cleaved from pentapeptide 4.15 with TBAF/THF in 70% yield, orwith mild HF/pyridine treatment in 90% yield to give 4.24 (Scheme 4.7).Removal of the Boc group from pentapeptide 4.14 with TFA, followed byneutralization of the TFA salt with NaHCO₃, gives a free amine which iscoupled with Boc-Abu-OH to give the desired hexapeptide 4.16 in 68%yield.

The Boc group is removed from hexapeptide 4.16, neutralized, and coupledwith the acetonide-protected MeLeu(3-OH) 4.6 to give heptapeptide 4.17in 58-80% yield. Acetonide-protected Meleu(3-OH) is formed by refluxingMeLeu(3-OH) in acetone overnight. Cleavage of the acetonide fromheptapeptide 4.17 with HCl/MeOH, followed by neutralization with NaHCO₃,gives the heptapeptide amine 4.18 in 58-80% yield. The heptapeptide 4.17is then coupled with 8-11 tetrapeptide under standard conditions to giveundecapeptide 4.19 in 32-50% yield (Scheme 4.8). After cyclization ofthe undecapeptide 4.19 with (PrPO₂)₃/DMAP, the CsA analog [MeLeu(3-OH)¹.D-MeSer(OH)³, Lys(2Cl-Cbz)⁷CsA 4.20 is obtained in 41-53% yield. Toallow coupling of the HIV protease inhibitor or another compound to theCsA analog, the Cbz group is cleaved with Pd(OH)₂/H₂ to yield thedesired free amine CsA analog 4.11 in quantitative yield.

Synthesis of η-Hydroxycyclosporin A (OL-17) from CsA

The semisynthetic cyclosporin A derivative, OL-17 is synthesized by theprocedure of Eberle and Nuninger (1992), J. Org. Chem. 57:2689 (Scheme4.9). Cyclosporin A is treated with acetylchloride and DMAP to yieldacetylcyclosporin A 4.21 in 65% yield. Allylic bromination with 1.2equivalent of NBS.AIBN(cat.) in CCl₄ at reflux gives allylic bromide4.22 which is used crude in the next reaction. As noted by Eberle andNuninger, the bromination reaction cannot be followed by TLC because thestarting material and product have identical R_(f) values. Thus, crudeallylic bromide 4.22 is dissolved in 2-butanone, treated withNMe₄OAc/cat.NaI, and the reaction is heated to 60° C. to giveη-acetoxyacetyl cyclosporin A 4.23 in 47% yield after purification byflash chromatography. The bisacetate 4.23 is treated with to NaOMe togive the final product, OL-17 2.11 in 50% yield.

Synthesis of CS-131 Acid Analog

The synthesis of CS-131 2.2, a truncated analog of the HIV proteaseinhibitor JG-365 2.1, follows the procedure of Rich et al., except thatthe original C-terminal methyl ester is replaced with an allyl ester.Incorporation of the allyl ester into the HIV protease inhibitor permitseither sanctification or palladium-mediated C-terminal deprotectionprior to coupling with CsA analog. The key step in the synthesis of theallyl ester analog of CS-131 involves reaction of the (2R,3S) Boc-Pheepoxide with the tripeptide Pro-Ile-Phe-allyl as shown in theretrosynthetic scheme (Scheme 5.1).

Synthesis of (2R,3S) Boc-Phe Epoxide 5.5

The key (2R,3S) Boc-Phe-epoxide is synthesized by following theprocedures of Thompson et al. (1992), J. Med. Chem. 35;1685 and Romeoand Rich (1994), Tetrahedron Lett. 35:4939 (Scheme 5.1.a, above).Reaction of Boc-Phe-OH 5.1 with N,O-dimethylhydroxylamine hydrochlorideand EDCI/HOBt produces the Weinreb amide 5.2 in 88% yield. Reduction ofthe Weinreb amide 5.2 with LiAlH₄ gives the aldehyde 5.3. The crudealdehyde 5.3 is converted via the Peterson olefination reaction to thealkene, which after reprotection of the amine withdi-tert-butylpyrocarbonate, gives the alkene 5.4 in 57% yield.Epoxidation of the alkene 5.4 with mCPBA under the conditions of Romeoand Rich gives the desired (2S,3R) epoxide 5.5 in 55% yield.

Synthesis of CS-131 Allyl Ester Analog 5.9

The tripeptide Boc-Phe-Ile-Phe-O-allyl 5.6 is synthesized in 56% overallyield using conventional EDCI/HOBt mediated peptide couplings (Scheme5.2). The tripeptide 5.6 is treated with HCl/dioxane and the resultingfree amine HCl salt 5.7 was refluxed with epoxide 5.5 to giveBoc-Phe-[HEA]-Pro-Ile-Phe-O-allyl 5.8 in 80% yield (Scheme 5.3).Cleavage of the Boc group in 5.8 with HCl/dioxane, and coupling of theresulting HCl salt with Cbz-Asn-OH to Phe-[HEA]-Pro-Ile-Phe-O-allyl withboth EDCI/HOBt and Cbz-Asn-OpNP consistently gives low yields of 5.9 andcomplex reaction products which were difficult ro purify. Activation ofCbz-Asn-OH can cause the formation of β-cyanoalanine, but additives,such as HOBt, are known to suppress these side reactions. Additionally,incorporation of asparagine into the inhibitor decreases the solubilityof the product in CH₂Cl₂, especially after removal of the allyl ester.

Synthesis of Trityl CS-131 Allyl Ester Analog 5.9 and CS-131 Acid 5.11

Cbz-Asn(Trt)-OH 5.10 is made using the conditions of Sieber and Riniker(Scheme 5.4). Coupling of Cbz-Asn(Trt)-OH withHCl-Phe-[HEA]-Pro-Ile-O-allyl gave 5.11 cleanly in 74% yield and waseasy to purify by flash chromatography. As hoped for, the solubilitycharacteristics of both ester 5.11 and acid 5.12 were greatly improvedand facilitated conversion to the completely deprotected CS-131derivative 5.13 (Scheme 5.5). To this end, ester 5.11 was saponifiedwith LiOH to give 5.12, which was treated with TPA/DCM to cleave thetrityl group and give completely deprotected inhibitor 5.13 in 88% yield(Scheme 5.5).

4-Hydroxypyran-2-one HIV Protease Inhibitor Synthesis

The retrosynthesis of the desired 4-hydroxypyrone inhibitor 2.4 is shownin Scheme 6.1. The inhibitor is constructed by reaction of a6-phenyl-4-hydroxy-pyran-2-one with2-tbutyl-4-hydroxy-benzenethiosulfonate under basic conditions inrefluxing ethanol. 2-tbutyl-4-hydroxy-benzenethiosulfonate is derivedfrom tert-butylhydroquinone via a Newman-Kwart Reaarangement followed byreaction with toluenesulfonyl bromide.

Synthesis of 2-t-butyl-4-t-butyldimethsilyhydroxy-benzenethiosulfonate6.5

The less hindered hydroxyl group in tert-butylhydroquinone 6.1 isselectively protected as the TBS ether by reaction with TBS-Cl to give6.2 in 88% yield (Scheme 6.1.a). Reaction of phenol 6.2 with NaH and N,N-dimethylthiocarbamoyl chloride produces the thiocarbonate 6.3 in 50%yield. Next, the Newman-Kwart rearrangement is performed by heatingoxygen-linked N,N-dimethylthiocarbamate 6.3 to 275-300° C. (in a sandbath) for approximately 20 minutes to give the thiol-lonked product 6.4in 50-70% yield (Scheme 6.2).

After rearrangement, the N,N-dimethylthiocarbamate 6.4 is reduced withLiAlH₄ to the free thiol, which is reacted with tosyl-bromide to givethe desired thiosulfonate 6.5 in 44% yield (Scheme 6.2). Reaction ofsimilar thiophenols with less reactive tosyl-chloride leads to disulfideformation, due to the unreacted thiophenol reacting with thethiosulfonate as it is produced.

Synthesis of Allyl Protected 4-hydroxypyran-2-one Inhibitor

Since the pyrone target compound 2.4 contains a carboxyl group forattaching to the CsA analog, t-butyl acetyl group was selected as aprotected linker for the inhibitor. Thus, the carboxyl group could beunmasked at tie end of the pyrone inhibitor synthesis for coupling tothe CsA analog.

The TBS group in 6.4 is cleaved with TBAF to give the phenol 6.9, whichis reacted with NaH and t-butyl chloroacetate/TBAI to form the ester6.10 in 97% yield (Scheme 6.6). However, attempts to saponify theN,N-dimethylthiocarbamate selectively over the t-butyl ester failed;only refluxing KOH/MeOH was able to cleave both the ester and the highlyhindered thiocarbamate to give 6.12 (Scheme 6.6). All other conditionsresulted in saponification of only the ester to give 6.11. The ease ofthe t-butyl ester saponification may be due to the small sterichindrance provided by the α-hydroxy phenyl group.

To circumvent this problem, allyl ether protection was chosen for pyroneinhibitor synthesis (Scheme 6.7). After cleavage of the TBS group, thecrude alcohol 6.9 is reacted with NaH/allyl iodide to give the allylether 6.13 in 85% overall yield. The thiocarbamate 6.13 is reduced withLiAlH₄ to give a free thiol, which is reacted crude with tosyl bromideto form the allyl protected thiosulfonate 6.14 in 44-75% yield. Finally,reaction of thiosulfonate 6.14 with pyrone 6.6, as previously described,smoothly yields the desired allyl-protected pyrone inhibitor 6.15 in 69%yield (Scheme 6.7).

Cleavage of Allyl Group From Pyrone Inhibitor 6.15

To attach the pyrone inhibitor 6.15 to a CsA derivative, the allyl etherhas to be cleaved and a linking functionality installed. To cleave theallyl group, a number of palladium mediated reactions conditions weretested, but only decomposition or no reaction was observed (Scheme 6.8).The use of SnBu₃H/THF/HOAc/Pd(PPh₃)₄ reaction conditions gives a 50%yield of phenol 6.16. By switching the reaction solvent to CH₂Cl₂, theyield of 6.16 was increased to 74%. Furthermore, by using Pd(OAc)₂ andPPh₃ to form Pd(0) in situ, the reaction proceeds more consistently.Na₂CO₃ extraction, followed by washing with ether, acidification of theaqueous phase with conc. HCl, and extraction with CH₂Cl₂ provides theproduct in sufficient purity (>95% by ¹HNMR) to take directly on to thenext reaction without further purification.

Synthesis of 4-hydroxypyran-2-one Ester Inhibitor 2.4

The last step of the pyrone inhibitor synthesis requires installation ofthe linking functionality. Phenol 6.16 is reacted with NaH/tert-butylchloracetate (cat.) at 0° C. followed by warming to RT overnight to givethe product 2.4, some decomposition products, and mostly startingmaterial (Scheme 6.9). Addition of tetrabutylammonium iodide (TBAI) tothe reaction mixture increases the rate of alkylation, but still only alow yield of pyrone ester 2.4 (20-30%) and significant amounts of a highrunning spot, presumed to be alkylation of the pyrone-OH, are obtained(Scheme 6.9).

The more robust protecting groups benzyl and allyl were evaluated(Scheme 6.10). The allyl-protected pyrone inhibitor 6.15 is reacted withCs₂CO₃/BnBr in NMP at 60° C. to give a 65% yield of the benzyl ether6.17. The allyl ether in 6.17 is cleaved to give the phenol derivative6.18, which was alkylated with Cs₂CO₃ at 60° C. to yield the benzylether pyrone ester 6.19 in 82% yield.

Alternatively, another approach takes advantage of the pyrone-OH acidity(Scheme 6.11). The pyrone hydroxyl proton in 6.16 is selectivelydeprotonated and alkylated with allyl iodide/DIEA give themono-protected allyl ether 6.20 in 48% yield. Alkylation of the phenol6.20 under the conditions previously described produces the ester 6.21in 82% yield. However, when allyl ether 6.21 is saponified, the product6.22 contained a methyl ether. This indicates that methoxide adds to thepyrone in 1,4 conjugate fashion, releasing allyl oxide (Scheme 6.12).Because of the extra steps and the poor overall yields, this protectinggroup “shuffle” is not preferred.

Since a one-step alkylation is the easiest route to the pyrone ester2.4, direct alkylation of the unprotected pyrone 6.16 with tbutylchloroacetate was employed. Reaction of 6.16 with tBuOK/tert-butylchloroacetate/TBAI at 0° C. warming to room temperature gives 2.4 in30-70% yield (Scheme 6.13). The potassium phenoxide anion reacts fasterwith tert-butyl chloroacetate/TBAU than does the sodium phenoxide anion,and also reacts faster than the pyrone anion, allowing better yields ofthe product 2.4. The best yields are obtained by keeping the reactiontemperature at 0° C. and carefully monitoring the reaction products byTLC.

Taking advantage of the ease of the ester hydrolysis, mild LiOH(aq)treatment cleanly produces the acid 6.23 in 78-85% yield. The amide 6.24was synthesized in 74% yield by reaction of acid 6.23 with mono-Bocprotected hexane diamine (Scheme 6.13).

Compound 6.25 can be synthesized using an identical approach. See alsoHagen et al. (1997), J. Med. Chem. 40:3707-3711.

VX-478 Analog Synthesis

Compound 2.6, a VX-478 analog modified to contain Tyr(OCH₂CO₂Et) in theP₁ poistion instead of a Phe, is synthesized from an appropriateepoxide, isobutylamine, and sulfonyl chloride (Scheme 7.1). In contrastto CS-131 2.2, which is derived from (2R,3S) Boc-Phe-epoxide, MES-14-0692.6 is derived from the (2S,3S)-Tyr-epoxide and thus requires adifferent synthesis than used previously for CS-131. An approach basedon a procedure reported by Kempf et al. (1995), Synlett 613 is used tosynthesize (S,S) Boc-Tyr(OBn)-epoxide, which was successfullytransformed into 2.6.

Synthesis of (2S,3S) Boc-Tyr(OH)-epoxide 7.12 via Felkin-Ahn DirectedAddition

L-Tyrosine 7.1 is perbenzylated with potassium carbonate/benzyl bromideto give ester 7.2, which was reduced with LiAlH₄ to give the aminoalcohol 7.3 in 73% yield after recrystallization from ethylacetate/hexane (Scheme 7. 1). Alcohol 7.3 is oxidized with pyridine-SO₃to aldehyde 7.4, which is reacted with chloromethyl lithium to give amixture of 7.5 (2S,3S) and 7.6 (2R,3S) epoxides in 89:11 ratio viaFelkin-Ahn directed addition. The chloromethyl lithium reagent used forthe aldehyde addition was derived from lithiation of bromochloromethanewith lithium metal. Since these epoxides are unstable to silica gel,crystallization of the HCl salts is required for purification. The crudeepoxides 7.5 and 7.6 are treated with HCl(aq)/THF to form thechlorohydrin HCl salts 7.7 and 7.8 which can be recrystallized at 5° C.to give a 30% yield of the pure (2S,3S) diastereomer 7.7 (Scheme 7.2).

HCl salt 7.9 is acylated with di-tert-butyl dicarbonate and thenepoxidized with KOH/MeOH to give Boc-Tyr(OH)-epoxide 7.12 in 25-50%yield (Scheme 7.4).

Synthesis of (2S,3S) Boc-Tyr(OBn)-epoxide 7.19 via chelation-controlledaddition

This synthesis was adapted for the synthesis of Boc-Tyr(OBN)-epoxide(Scheme 7.5). Boc-Tyr(OBn)-OH 7.13 is reacted with Cs₂CO₃MeI to form themethyl ester 7.14. The crude ester 7.14 is reduced to the aldehyde withDIBAL and treated with vinylmagnesium bromide in situ to give ca. 4:1ration of (3S,4S) to (3R,4S) allylic alcohols 7.15 and 7.16, which areseparable by column chromatography. The (3S,4S) allylic alcohol 7.15 istreated with mesyl chloride to give 7.17 (Scheme 7.6). Crude mesylate7.17 is ozonolyzed to the adlehyde, which is reduced with NaBH₄ to givealcohol 7.18 in excellent yield. Reaction of 7.18 with NaH in refluxingTHF smoothly produces (2S,3S) Boc-Tyr(OBn)-epoxide 7.19 in 86% yield.

Completion of Compound 2.6

The hydroxyethylamine isostere is constructed by reaction of the epoxide7.19 with isobutylamine (Scheme 7.7). After stirring overnight inmethanol, the reaction mixture is concentrated from ether several timesand then hydrogenated over Pd(OH)₂/methanol to give the fullydeprotected hydroxymethylamine 7.20 in 85% yield. Although 7.20 can berecrystallized from ethyl acetate/hexane, it is generally moreconvenient to use the crude product directly in the next reaction. Tothe fully deprotected hydroxyethylamine 7.20 in CH₂Cl₂ at 0° C., wasadded 4-carbobenzyloxyamino-phenylsulfonyl chloride dropwise. Thisyields two products; a very polar spot 7.21 in 40% yield, and a highrunning spot 7.22 in 35% yield. The products correspond tomono-sulfonylation of the phenol 7.21 and di-sulfonylation of the phenoland amine 7.22, indicating that the phenol reacts preferentially overthe amine.

Protection of the phenol before sulfonylation of the amine isaccomplished by treating hydroxyethylamine 7.20 with 2 equivalents ofimidazole and 2 equivalents of TBS-Cl (Scheme 7.8). TLC analysis showedformation of an intermediate, followed by formation of a higher runningspot, which suggested that the TBS-Cl was reacting with both the freeamine and the phenol. After aqueous workup which presumably cleaves theN-TBS bond, treatment of the crude OTBS-protected intermediate with4-carbobenzylaminophenylsulfonyl chloride/TEA in THF smoothly producesthe desired N-sulfonylated/O-TBS protected product 7.23 in 61% yield(Scheme 7.8). The TBS group is cleaved with HF/pyridine to give thephenol, which is reacted directly with Cs₂CO₃/ethyl bromoacetate indioxane at 40-50° C. for 3 hours to form the ester 7.24 in 71% overallyield. The ester 7.24 is reacted with HCl/dioxane to give the HCl salt,which is treated with 3-(S)-hydroxytetrahydrofuran p-nitrophenylcarbonate/TEA to give Cbz-protected analog 7.5 (Scheme 7.9). At thispoint, the ester can be hydrolyzed to give the free acid 7.26 or it canbe hydrogenated with Pd(OH)₂ to give 2.6 in quantitative yield.

Synthesis of Conjugates 2.8, 2.10, and 2.12

(MeLeu(3-OH)¹, D-MeSer³, Lys(2CL-Cbz)⁷)CsA 4.20 (Scheme 4.9) ishydrogenated using Pd(OH)₂/MeOH to give the free amine 4.11, which wasused in the next reaction without further purification. The completelydeprotected CS-131 acid derivative 5.13 is coupled to the CsA analogfree amine 4.11 with PyAOP to give the desired conjugate 2.8 in 50%yield (Scheme 8.2).

Similarly, the pyrone inhibitor 6.23 is coupled to the CsA analog freeamine with PyAOP to give the conjugate 2.9 in 56% yield (Scheme 8,3),and to Cbz protected 7.26 to give the Cbz-protected conjugate 8.2 in 50%yield. Cleavage of the Cbz group from 8.2 produces the fully deprotectedconjugate 2.10 (Scheme 8.4).

To provide a reactive group for coupling of the HIV protease inhibitors,them semisynthetic derivative OL-17, 2.11 is reacted with p-nitrophenolchloroformate to produce the carbonate 8.3 in 74% yield (Scheme 8.5).After cleavage of the Boc group in pyrone inhibitor 6.24, the resultingHCl salt was reacted with the carbonate 8.3 in THF to give thesemisynthetic conjugate 2.12 in 24% yield.

Conjugate 2.8 (Scheme 12.1), which incorporates (MeLeu(3-OH)¹, D-MeSer³,Lys⁷)CsA and the hydroxyethylamine HIV protease inhibitor 5.13, exhibitspotent activity against HIV protease (K_(i)=1 nM) and againstcyclophilin (K_(i)=20 nM). This result clearly shows that each domain inthe conjugate retains the ability to independently inhibit each enzyme.The conjugate also binds to both enzymes simultaneously: preincubatingthe conjugate with a 1000-fold excess of cyclophilin did not alter theHIV protease inhibitory activity of the conjugate. Additionally, apreliminary ultracentrifugation experiment indicated that a complex (30KDa) which was larger than either enzyme (18 and 22 KDa) had beenformed, which corroborates the initial data that the conjugate bindssimultaneously to cyclophilin and HIV protease.

The pyrone conjugate 2.9 (Scheme 12.2) also strongly inhibits bothcyclophilin (K_(i)=9.3 nM) and HIV protease (K_(i)=3.4 nM). Since theparent Parke-Davis HIV protease inhibitor has a K_(i)=7.3 nM against HIVprotease, the acetoxy group/lysine side-chain has no effect on theconjugate's ability to inhibit HIV protease.

Design, Synthesis and Biological Activity of the Conjugate Containingthe Acetoxy Acid-Modified VX-478 inhibitor 7.26

VS-478 2.5 (Scheme 12.3) has excellent bioavailability and antiviralactivity, and thus provided an inhibitor with a different cellpenetration profile than those used for conjugates 2.8 and 2.9. In thedesign and synthesis of conjugates 2.9 and 2.9, the C-terminus of theknown inhibitor CS-131 and the corresponding “C-terminus” end of thepyrone inhibitor are available for functionalization. In 2.5, theC-terminal position corresponds to the aniline nitrogen of thesulfonamide group. However, the x-ray structure of VS-478 complexed withHIV protease indicates that this nitrogen is hydrogen bonded to theenzyme and sterically constrained, so much so that derivatization of theaniline nitrogen would lead to unfavorable interactions with the enzyme.In contrast, the para position of the benzyl group in the P₁ position isexposed to solvent and therefore used as the functionalization position.An acetoxy group was chosen to provide the linking functionality as inconjugate 2.9. The coupling of the modified VS-478 inhibitor 7.26 to theCsA analog 4.11 proceeds in a straight-forward manner, to give thedesired conjugate 2.10 (Scheme 12.3). Notably, inhibitor 7.26 wassoluble in DCM which was used as the coupling solvent.

Compound 2.6, which is modified VX-478 inhibitor containing an ethylacetoxy linking group, shows approximately the same activity against HIVprotease (K_(i)=1 nM) as VS-478 2.5 (K_(i)=0.6 nM), thereby establishingthat modification of VX-478's P₁ substituent has no effect on itsinhibition of HIV protease. The conjugate 2.10 (Scheme 12.2) alsoexhibits potent activity against both cyclophilin (K_(i)=6 nM) and HIVprotease (K_(i)=2.3 nM), indicating that the CsA analog has no effect onthe inhibition of HIV protease.

Conjugates 2.8, 2.10, and 2.12 have been evaluated for anti-HIV activityin infected cell lines against both susceptible and protease-resistantHIV; see the Examples.

Treatment of HIV-Mediated Disorders using Pharmaceutical CompositionsContaining the Subject Compounds

The invention includes a method of inhibiting or treating HIV-mediateddisorders in mammals (especially humans). The method includesadministering to a subject in need thereof an effective HIV-replicationinhibiting amount of one or more of the subject compounds. The compoundsmay be administered neat or in the form of a pharmaceutical compositioncomprising one or more active ingredients in combination with apharmaceutically-acceptable carrier.

In mammalian subjects, the compounds of Formula I can be administeredorally, parenterally (including subcutaneous, intradermal, intramuscularand intravenous injection), rectally, and topically (including dermal,buccal, and sublingual administration) in combination with an inertliquid or solid pharmaceutically-acceptable carrier which is suitablefor the method of administration chosen. The carrier must bepharmaceutically acceptable in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient thereof. Such pharmaceutical carriers are well known in theart. The preferred route of administration is orally.

In in vitro applications, such as in the study of mutant cell types,virus types, or other cellular investigations, the pharmaceuticalcompositions of the present invention are preferably administered to thecells by adding a predefined amount of a compound of Formula I, dilutedin a suitable diluent, to the cell culture medium. As used herein, theterms “administering” or “administration” are synonymous with “treating”or “treatment.” In essence, administering to cells in vitro one or moreof the compounds of Formula I entails contacting the cells with thecompounds or salts of the compounds.

The in vivo dosage in humans and other mammals depends largely upon theaffliction being treated, the time since onset of the condition, theprogression of the disease, and the age and general health of thepatient being treated. Determining the optimum dosage for any givenpatient is essentially an empirical and ongoing process. Inhibition orprevention of HIV-mediated disorders in infants and children who arediagnosed early in the progression of the condition may optimallyrequire a more (or less) aggressive treatment than in older patients inmore terminal stages of AIDS. Of primary importance in optimizing themost effective dosage is that each patient be carefully monitoredthroughout the course treatment to follow the progression, if any, ofthe condition.

A suitable effective dose for most conditions ranges from about 1 mg/kgbody weight to about 2 g/kg body weight per day, and is preferably inthe range of from about 5 to about 500 mg/kg body weight per day(calculated as the non-salt form of the Formula I compound). The totaldaily dose may be given as a single dose, multiple doses, e.g., two tosix times per day, or by intravenous infusion for a selected duration.Dosages above or below the above-cited ranges are within the scope ofthe invention and such dosages may be administered to individualpatients if the circumstances so dictate.

For example, in a 75 kg mammal, a typical daily dosage might fall withinthe range of from about 75 mg to about 7.5 g per day. If discretemultiple doses are indicated, treatment might typically comprise 4 equalfractional doses given at 8 hour intervals to supply the total dailydosage.

The active ingredients used in the above-described method are analogsand conjugates of CsA and pharmaceutically-acceptable salts thereof. Alloptical, geometric, and positional isomers of the compounds of FormulaI, including racemic mixtures or pure or enriched enantiomeric forms,geometric isomers, and mixtures thereof, are within the scope of thisinvention.

By the term “pharmaceutically-acceptable salt” is meant any saltconventionally used in the formulation and administration ofpharmaceutical preparations. This term encompasses inorganic salts suchas nitrates, phosphates, sulfates, and chlorides, as well as mono anddi-substituted basic salts of sodium, potassium, calcium, and the like.Organic salts such as malonates, fumarate, succinates, crotonates, andthe like are also encompassed by the term “pharmaceutically-acceptablesalt.” The foregoing list is exemplary, not exclusive. A large number ofsalts acceptable for pharmaceutical administration are known to those ofskill in the art.

The pharmaceutical composition may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. The term “unit dosage” or “unit dose” means apredetermined amount of the active ingredient sufficient to be effectivefor treating HIV-mediated disorders in man. All methods include the stepof bringing the active compound(s) into association with a carrier andone or more optional accessory ingredients. In general, the formulationsare prepared by uniformly and intimately bringing the active compoundinto association with a liquid or solid carrier and then, if necessary,shaping the product into desired unit dosage form.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,boluses or lozenges, each containing a predetermined amount of theactive compound; as a powder or granules; or in liquid form, e.g., as anaqueous solution, suspension, syrup, elixir, emulsion, dispersion, orthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets may be made by molding in asuitable machine a mixture of the powdered active compound with anysuitable carrier.

Formulation suitable for parenteral administration conveniently comprisea sterile preparation of the active compound in, for example, water forinjection, saline, a polyethylene glycol solution and the like, which ispreferably isotonic with the blood of the recipient.

Useful formulations also comprise concentrated solutions or solidscontaining the compound(s) of Formula I which upon dilution with anappropriate solvent give a solution suitable for parenteraladministration.

Preparations for topical or local applications comprise aerosol sprays,lotions, gels, ointments, suppositories etc., andpharmaceutically-acceptable vehicles therefore such as water, saline,lower aliphatic alcohols, polyglycerols such as glycerol, polyethyleneglycerol, esters of fatty acids, oils and fats, silicones, and otherconventional topical carriers. In topical formulations, the compounds ofFormula I are preferable utilized at concentration of from about 0.1% to5.0% by weight.

Compositions suitable for rectal administration comprise a suppository,preferably bullet-shaped, containing the active ingredient andpharmaceutically acceptable vehicles therefore, such as hard fat,hydrogenated cocoglyceride, polyethylene glycol, and the like. Insuppository formulations, the compounds of Formula (1) are preferablyutilized at concentrations of from about 0.1% to 10% by weight.

Compositions suitable for rectal administration further comprise arectal enema unit containing the active ingredient and pharmaceuticallyacceptable vehicles therefore such as, for example, 50% aqueous ethanolor an aqueous salt solution which is physiologically compatible with therectum or colon. The rectal enema unit comprises an applicator tipprotected by an inert cover, preferably comprised of polyethylene,lubricated with a lubricant such as white petrolatum and preferablyprotected by a one-way valve to prevent back-flow of the dispensedformula, and of sufficient length, preferably two inches, to be insertedinto the colon via the anus. In rectal formulations, the compounds ofFormula (1) are preferably utilized at concentrations of from about 5.0%to 10% by weight. Useful formulations also comprise concentratedsolutions or solids containing the active ingredient which upon dilutionwith an appropriate solvent, preferably saline, give a solution suitablefor rectal administration. The rectal compositions include aqueous andnon-aqueous formulations which may contain conventional adjuvants suchas buffers, bacteriostats, sugars, thickening agents and the like. Thecompositions may be presented in rectal single dose or multi-dosecontainers.

In pharmaceutical compositions suitable for administration byinhalation, the active ingredient(s) is combined with a carriercomprising a solid in a micronized powder having a particle size in therange of about 5 microns or less to about 500 microns, or a liquidcarrier, for rapid inhalation through the oral passage from aconventional metered-dose inhaler or nebulizer. Suitable liquid nasalcompositions include conventional nasal sprays, nasal drops and thelike, of aqueous solutions of the active ingredient and optionaladjuvants.

In addition to the aforementioned ingredients, the pharmaceuticalformulations of this invention may further include one or more optionalaccessory ingredient(s) utilized in the art of pharmaceuticalformulations, i.e., diluents, buffers, flavoring agents, colorants,binders, surface active agents, thickeners, lubricants, suspendingagents, preservatives (including antioxidants) and the like.

EXAMPLES

The following Examples are included solely to aid in a more completeunderstanding of the invention disclosed and claimed herein. TheExamples do not limit the scope or utility of the invention in anyfashion.

General Experimental Procedures

General Procedure A. BOP-Cl Couplings

A solution of the N-protected amino acid (1.1 eq) and amino acid esteror peptide amino acid ester (1.0 eq) was cooled to 0° C. in DCM (0.15M). To the cooled solution was added TEA (2.1 eq) and then BOP-Cl (1.1eq) in one portion. (An extra equivalent of TEA was added if the aminoacid ester or peptide ester was in the form of an HCl salt). The cloudysolution was stirred overnight, warming to room temperature, at whichpoint the solution became clear. The reaction was poured into ethylacetate (3×reaction volume) and washed with KHSO₄, H₂O, NaHCO₃ and thenbrine. After drying over Na₂SO₄ and filtering, the filtrate wasconcentrated in vacuo to an oil and purified by flash chromatography.

General Procedure B. BOP-Cl Couplings via Pre-activation

A solution of the N-protected amino acid (1.1 eq) was cooled to 0° C. inDCM (0.15 M). TEA (1.1 eq) and BOP-Cl (1.1 eq) were added and thereaction was stirred at 0° C. for one hour. A solution of the amino acidester or peptide ester HCl salt and TEA (1.1 eq) in ca. 1 M DCM was thenadded to the reaction. After stirring overnight and allowing thereaction to warm to room temperature, the reaction was poured into ethylacetate (3×reaction volume) and washed with KHSO₄, H₂O, NaHCO₃, and thenbrine. After drying over Na₂SO₄ and filtering, the filtrate wasconcentrated in vacuo to an oil and purified by flash silica gelchromatography.

General Procedure C. EDCI/HOBt Peptide Coupling

A solution of the N-protected amino acid (1.1 eq) and amino acid esterHCl salt (or peptide amino acid ester HCl salt) (1.0 eq) in DCM or DMF(0.20 M) was cooled to 0° C. and treated with TEA (1.05 eq), HOBt (1.5eq), and EDCI (1.1 eq) in one portion. The solution was stirredovernight while warming to room temperature, poured into ethyl acetate(3×reaction volume) and washed with KHSO₄, H₂O, NaHCO₃, and then brine.After drying over Na₂SO₄ and filtering, the filtrate was concentrated invacuo to an oil and purified by flash silica gel chromatography.

General Procedure D. Boc Group Cleavage with 4N HCl/Dioxane

The Boc-protected amine was dissolved in 4 N HCl/dioxane (20-100 eq) atroom temperature and stirred until TLC showed consumption of startingmaterial (ca. 1 hr). The reaction was concentrated in vacuo and thenconcentrated from ether (3×) and DCM (3×) to produce a white solid.

General Procedure E. TFA Cleavage of Boc Groups

A solution of the Boc-protected amine in DCM (0.2 M) was cooled to −15°C. in a MeOH/ice bath and treated with TFA, bringing the totalconcentration of the reaction to 0.1M. The reaction was stirred in thecold, until TLC showed consumption of starting material (ca. 1-2 hr),and then added dropwise into a slurry of NaHCO₃ (1.1 g per ml of TFA) inH₂O and DCM. The phases were separated and the aqueous phase wasextracted with DCM (3×). The organic layers were combined, dried overNa₂SO₄, filtered, and concentrated in vacuo to give the neutral freeamine.

General Procedure F. Acetonide Protection of MeLeu(3-OH)

2S,3R MeLeu(3-OH) 3.6 was refluxed overnight in freshly distilledacetone (0.003 M) until an almost clear solution of acetonide-protectedMeLeu(3-OH) 4.6 was obtained. After concentrating the reaction volume to1.5-5.0 ml, the amino acid was added directly to the coupling reactiondescribed in general procedure G.

General Procedure G. Synthesis of CsA Heptapeptide byAcetonide-Protected MeLeu(3-OH)

To solution of the hexapeptide free amine (1.0 eq), N-methylmorpholine(1.1 eq), and HOBt (2.2 eq) in THF (0.05M) was added acetonide-protectedMeLeu(3-OH) 4.6 (1.1 eq). The mixture was cooled to 0° C. and DCC (1.1eq) was added in one portion. After stirring the reaction overnight, thedicyclohexylurea (DCU) that precipitated was removed by filtrationthrough celite and washed with small portions of DCM (3×). The filtratewas concentrated in vacuo and dissolved in ethyl acetate whichprecipitated additional DCU that was filtered off as before. Thefiltrate was concentrated in vacua and the residue was purified by flashchromatography using acetone/hexane gradients.

General Procedure H. Cleavage of N,O-Isopropylidene fromAcetonide-Protected Heptapeptide

To a solution of the N,O-isopropylidene-protected peptide in methanol(0.05 M) was added 1.0 N HCl (aq) (4.0 eq) and the reaction was stirredfor 12 hours at room temperature. NaHCO₃ (12 eq) was added and thereaction was concentrated in vacuo. The resulting white slurry was takenup in 2-4% MeOH/DCM and purified by flash chromatography with 2-4%MeOH/DCM to yield a white foam.

General Procedure I. CsA Linear Undecapeptide Synthesis via “7+4”Coupling

To a solution of the amine heptapeptide benzyl ester (1.0 eq) andN-protected tetrapeptide acid (1.3 eq) in DCM (0.05 M) was added BOPreagent (1.3 eq) and N-methylmorpholine methylmorpholine (2.0 eq). Thereaction was stirred for 3 days at room temperature and thenconcentrated in vacuo. The residue was dissolved in DCM and washed withH₂O, the phases were separated, and the aqueous layer was washed withDCM (2×). The organic layers were combined and dried over Na₂SO₄,filtered, concentrated in vacuo, and purified by flash chromatographyusing MeOH/DCM mixtures as the eluant.

General Procedure J. Cyclization of Undecapeptide to Form Cyclosporin AAnalogs

A solution of the undecapeptide in ethanol at 0° C. was treated with 0.2N NaOH. The reaction was stirred at 0° C. for 1.5 hours, at which pointan additional 1.0 eq of 0.2 N NaOH was added and the stirring wascontinued at 0° C. for 6-10 more hours. After acidification with 0.2 NHCl to pH 6, the solution was diluted with brine and extracted with DCM(4×), the organic layers were combined, dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was dissolved in DCM andpropylphosphonic anhydride (50% w/v in DCM) and DMAP were added. Themixture was stirred at room temperature for 3 days under argon,concentrated in vacuo, and purified by flash chromatography usingacetone/hexanes as the eluant to yield a white foam.

General Procedure K. Cleavage of the Cbz Group from the 7-Position ofthe CsA Analogs

A round bottom flask containing the CsA analog and Pd(OH)₂ (10-50% byweight) were thoroughly flushed with argon and dissolved in methanol(1-2 ml). The flask was then evacuated and the vacuum was broken withhydrogen (repeated 3×). After stirring for 1-3 hours under I atm of H₂,the mixture was filtered through an acrodisk and the filtrate wasconcentrated in vacuo to give the free amine CsA analog which was useddirectly in the next reaction.

Example 1 Synthesis of 2S,3R MeLeu(3-OH)

Route 1

Syn-(4S,2′S,3′R)-3-(4′-methyl-3′-hydroxy-2′-bromo-1′-pentanoyl)4-benzyl-2-oxazolidinone 3.8

A solution of (4S)-3-bromoacetyl-4-phenyl-2-oxazolidinone 3.7 (1.7 g.5.7 mmol) in ether (28 ml) was cooled to −78° C., and treated with TEA(1.11 ml, 7.98 mmol), followed by di-n-butylboron triflate (1.73 ml,6.04 mmol). The cooling bath was removed and the reaction was stirred atroom temperature for 1.5 hours. After cooling the reaction back to −78°C. with vigorous stirring, isobutyrlaldehyde (0.545 ml, 5.91 mmol) wasadded and the resulting reaction mixture was stirred at −78° C. for 0.5hours and at 0° C. for 2 hours. The reaction was diluted with ether (50ml), washed with KHSO₄ (2X), and concentrated in vacuo. The residue wasbrought up in 1:1 MeOH/H₂O (20 ml), cooled to 0° C., followed byaddition of 30% H₂O₂ (7 ml). After stirring the reaction for 1 hour at0° C., it was concentrated in vacuo to give a residue that was dilutedwith ether (50 ml) and washed with H₂O, 1 N NaHCO₃, brine, dried overNa₂SO₄, filtered and concentrated in vacuo. The residue was purified byflash chromatography (25-30-35% EthOAc/hexane) to give 919 mgs (44%yield) of the aldol product as a foam. R_(f)=0.32 (30% EthOAc/hexane).[α]_(D) ²³ −+51.9 (c 0.30, CDCl₃), ′H NMR (300 MHz, CDCl₃) δ7.42-7.18(m, 5H), 5.91 (d, 1H, J=2.3 Hz), 4.8-4.65 (m, 1H), 4.31-4.19 (m, 2H),3.51 (dd, 1H, J=3, 7.6 Hz), 3.32 (dd, 1H, J=13.5, 3.3 Hz), 2.81 (dd, 1H,J=9.5, 13.5 Hz), 1.96-1.83 (m, 1H), 1.07 (d, 3H, J=6.7 Hz), 0.97 (d, 1H,J=6.7 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ169.55, 152.37, 134.61, 129.47,129.08, 127.56, 75.59, 66.35, 55.24, 48.93, 36.99, 31.96, 18.78, 18.23.HR-EI: calculated for C₁₆H₂₀NO₄Br 369.0576, found 369.0585.

Syn-(4S,2′S,3′R)-3-(4′-methyl-3′-O-N-methylcarbamoyl-2′-bromo-1′-pentanoyl)-4-benzyl-2-oxazolidinone3.9

A solution of the aldol product 3.8 (694 mg, 1.87 mmol) in toluene (6.5ml) at room temperature was treated with N-methylisocyanate (0.551 ml,9.35 mmol) and BF₃-OEt₂ (0.276 ml, 2.24 mmol). The reaction was stirredfor 1 hour, quenched with 5% NaHCO₃ (6 ml) and stirred for an additional30 minutes. After diluting with H₂O (25 ml) and extracting with DCM(3×25 ml), the organic phases were combined, dried over Na₂SO₄,filtered, and concentrated in vacuo. The residue was purified by flashchromatography (25-30-35% EthOAc/hexane) to give 583 mgs (73% yield) ofthe carbamate as a white foam. R_(f)=0.20 (30% EthOAc/hexane). [α]_(D)²³=+55.6 (c 0.67, CDCl₃), ¹H NMR (300 MHz, CDCl₃) δ7.34-7.19 (m, 5H),6.05 (d, 1H, J=3.1 Hz), 4.91-4.78 (m, 1H), 4.64-4.54 (m, 1H), 4.35 (m,1H), 4.20 (dd, 1H, J=8.9, 2.5 Hz), 3.31 (dd, 1H, J=13.4, 3.3 Hz),2.90-2.73 (m, 4H), 2.15-2.04 (m, 1H), 1.02 (d, 3H, J=6.8 Hz), 0.95 (d,3H, J=6.6 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ166.69, 156.66, 153.49,135.10, 129.47, 128.10, 127.39, 76.62, 66.81, 56.35, 50.7, 37.56, 31.41,27.67, 18.29, 18.22. HR-EI: calculated for C₁₈H₂₃N₂O₅Br 428.0771, found428.0777.

(4R,5R)-3-N-Methyl-4-methylester-5-isopropyl-2-oxazolidinone 3.11

A solution of oxazolidinone carbamate 3.9 (481 mg, 1.12 mmol) in 3:1THF:H₂O (12 ml) was cooled to 0° C. and treated with LiOH (2.24 ml, 1 NLiOH). The reaction was stirred for 30 minutes and concentrated in vacuoto give a residue that was dissolved in H₂O and wathed with EthOAc (3×50ml), acidified to pH 2 with 2 N HCl, and extracted with DCM (3×75 ml).The organic layers were combined, dried over Na₂SO₄, filtered, andconcentrated in vacuo to give 288 mgs (82% yield) of the acid 3.10,which was used crude in the next reaction.

A solution of 3.10 (236 mg, 0.88 mmol) in DMF (6 ml) at room temperaturewas treated with tert-butoxide (494 mg, 4.4 mmol) After stirring thereaction for 30 minutes, toluene was added, and the reaction wasconcentrated in vacuo to give a residue that was dissolved in H₂O (50ml), washed with EthOAc, acidified to pH 2 with 2 N HCl, and extractedwith EthOAc (6×50 ml). The organic layers were combined, dried overNa₂SO₄, filtered, and concentrated in vacuo. The residue was dissolvedin 3:1 THF/MeOH (8.8 ml) and treated with TMS-diazomethane (0.88 ml, 2 Min hexane). The reaction was stirred for 1 hour and concentrated invacuo to a residue which was purified by flash chromatography (30-40%EthOAc/hexane) to give 128 mgs (72% over two steps) of 3.11 as a clear,fluid-like oil. R_(f)=0.25 (40% EthOAc/hexane). [α]_(D) ²³=−2.7 (c 0.38,CDCl₃), ¹H NMR (300 MHz, CDCl₃) δ4.26 (d, 1H, J=7.6 Hz), 4.19 (dd, 1H,J=9.1, 7.6 Hz), 3.82 (s, 3H), 2.85 (s, 3H), 1.86-1.71 (m, 1H), 1.07 (d,3H, J=6.5 Hz), 0.98 (d, 3H, J=6.6 Hz). ¹³C NMR (75.5 MHz, CDCl₃)δ168.89, 81.04, 63.81, 52.49, 42.08, 30.25, 29.3, 18.99, 18.44. HR-EI:calculated for C₉H₁₅NO₄ 201.1001, found 201.0992.

(2S,3R)-MeLeu(3-OH) 3.6

The oxazolidinone methyl ester 3.11 (400 mg, 2.0 mmol) was dissolved inethanol (2.27 ml) and treated with KOH (2.27 ml, 1.03 N KOH). Thereaction was refluxed for 1 hour, cooled to room temperature, andconcentrated in vacuo to give trade 3.12. The resulting white slurry wasdirectly treated with KOH (aq) (4.4 ml, 1.59 M), stirred at 80° C.overnight, and concentrated in vacuo. After acidifying the mixture to pH6 with 1 N HCl, it was purified by ion-exchange chromatography (“DOWEX”50×, 4% NH₄OH) to give 242 mgs (75% yield) of 3.6 as a white solid. ¹HNMR (300 MHz, D₂O) δ4.8 (s, 3H), 3.69 (dd, 1H, J=7.15, 5.0 Hz), 3.56 (d,1H, J=7.15 Hz), 2.73 (s, 3H), 1.82 (m, 1H), 0.97 (d, 3H, J=3.8 Hz), 0.95(d, 1H, J=3.66 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ167.98, 80.28, 63.02,51.72, 29.47, 28.52, 18.21, 17.63.

(2S,3R) MeLeu(3-OH) Synthesis

Route 2

(2E)-4-Methyl-2-penten-1-ol 3.14

A solution of 3.13 (13 g, 112.5 mmol) in THF (300 ml) at −78° C. wastreated dropwise with DIBAL (248 ml of a 1 M solution in THF). Thereaction was stirred for 2 hours at −78° C., quenched carefully withMeOH, and diluted with ether (200 ml). The mixture was washed with 3 NHCl, phases were separated, and the aqueous phase was extracted withether (3×100 ml). The combined organic layers were washed with brine,dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue wasdistilled under reduced pressure to give 9.6 g (85% yield) of thealcohol 3.14. R_(f)=0.55 (30% EthOAc/hexanes). ¹H NMR (300 MHz, CDCl₃)δ5.73-5.5 (m, 1H), 4.12-4.03 (m, 1H), 2.39-2.2 (m, 1H), 1.55 (bs, 1H),1.0 (d, 6H, J=6.6 Hz).

(2S,3R)-4-Methyl-2,3-epoxy-1-ol 3.15

A flame-dried flask was charged with 4-angstrom powdered molecularsieves (2.34 g) and DCM (275 ml) and then cooled to −20° C. in MeOH/icebath. D-(−)-diethyl tartarate (0.99 ml, 4.7 mmol) and Ti(Oi—Pr)₄ (1.16ml, 3.9 mmol) were added sequentially, followed by cumene hydroperoxide(80% solution, stored over 4-angstrom molecular sieves, 28.9 ml, 156mmol) at a moderate rate. The resulting reaction mixture was transferredto the refrigeration apparatus set at −20° C., stirred for 30 minutes,and then treated with allylic alcohol 3.14 (7.8 g, 78 mmol) dropwiseover 10 minutes, and the resulting mixture was stirred overnight at −20°C.

A freshly prepared solution of ferrous sulfate heptahydrate (25.8 g) andtartaric acid (7.8 g) in deionized H₂O (78 ml) was cooled to 0° C. Theepoxidation reaction was allowed to warm to 0° C. and was then pouredinto a beaker containing the precooled ferrous sulfate solution. Thetwo-phase mixture was stirred for 5-10 minutes and then transferred to aseparatory funnel. After the phases were separated, the aqueous phasewas extracted with ether (2×100 ml) and the combined organic layers weretreated with a precooled solution of 30% NaOH (w/v) (7.8 ml) insaturated brine and stirred vigorously for 1 hour at 0° C. The reactionmixture was diluted with H₂O, the phases were sepated, and the aqueouslayer was extracted with ether (3×100 ml). The combined organic phaseswere dried over Na₂SO₄, filtered, and concentrated in vacuo to give aresidue which was purified by flash chromatography (2:1 hexane/EthOAc)to give 6.5 g (72% yield) of the epoxide 3.15 as a colorless oil.R_(f)=0.35 (33% EthOAc/hexanes). ¹H NMR (300 MHz, CDCl₃) δ3.92 (d, 1H,J=11.7 Hz), 3.72-3.53 (m, 1H), 3.02-2.95 (m, 1H), 2.76 (dd, 1H, J=6.8,2.4 Hz), 2.30 (m, 1H), 1.66-1.50 (m, 1H), 1.03 (d, 3H, J=6.7 Hz), 0.97(d, 3H, J=6.9 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ61.94, 61.22, 57.59,30.03, 18.96, 18.31.

(25,3R)-4-Methyl-2,3-epoxy-1-O-methylcarbamate 3.17

A solution of the epoxy alcohol 3.15 (1.014 g, 8.75 mmol) in DCM (130ml) was treated sequentially with TEA (3.05 ml, 21.88 mmol) andmethylisocyanate (1.032 ml, 17.5 mmol). The mixture was stirred underargon for 20 hours, quenched with satd. NH₄Cl, and transferred to aseparatory funnel. The phases were separated and the aqueous phase wasextracted with DCM (3×100 ml). The combined organic phases were driedover Na₂SO₄, filtered, and concentrated in vacuo to give a residue whichwas purified by flash chromatography (30% EthOAc/hexane) to give 1.32 g(87% yield) of the carbamate 3.17 as a colorless oil. R_(f)=0.55 (30%EthOAc/hexanes). ¹H NMR (300 MHz, CDCl₃) δ4.75 (bs, 1H), 4.40 (dd, 1H,J=12.21, 2.85 Hz), 3.89 (dd, 1H, J=12.13, 6.4 Hz), 3.01 (m, 1H), 2.81(d, 3H, J=4.94 Hz), 2.65 (dd, 1H, J=6.72, 2.67 Hz), 1.65-1.50 (m, 1H),1.01 (d, 3H, J=6.72 Hz), 0.96 (d, 3H, J=6.87 Hz). ¹³C NMR (75.5 MHz,CDCl₃) δ65.63, 61.92, 54.79, 30.07, 27.50, 18.87, 18.25. FABMS: found174.1 [M+H⁺].

(4R,5R)-3-N-Methyl-4-hydroxymethyl-5-isopropyl-2-oxazolidinone 3.16

A solution of KH (224 mg, 5.6 mmol) in THF (50 ml) at 0° C. was treateddropwise with a solution of the carbamate 3.17 (647 mg, 3.74 mmol) inTHF (5 ml). The reaction was stirred at 0° C. for 2 hours and then atroom temperature for 1 hour. After quenching the reaction at 0° C. with1 N KHSO₄ to pH 5, it was extracted with CHCl₃ (9×50 ml) and thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. The residue was purified by flash chromatography(30-40% acetone/hexane) to give 415 mgs (65% yield) of the desiredoxazolidinone 3.16 as a white solid, and 104 mgs (16% yield) of theother isomer 3.18. Data for 3.16; R_(f)=0.38 (50% acetone/hexanes). ¹HNMR (300 MHz, CDCl₃) δ4.05 (dd, 1H, J=10.38, 7.04 Hz), 3.86 (m, 1H),3.61 (m, 1H), 2.94 (s, 3H), 2.86 (m, 1H), 2.15 (m, 1H), 1.09 (d, 3H,J=6.45 Hz), 0.956 (d, 3H, J=6.6 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ159.38,82.66, 61.08, 52.28, 29.80, 27.49, 19.68, 18.94. HR-EI: calculated forC₈H₁₅NO₃ 173.1052, found 173.1057. Data for 3.18; R_(f)=0.45 (50%acetone/hexanes). ¹H NMR (300 MHz, CDCl₃) δ4.32 (dd, 1H, J=8.5, 6.9 Hz),4.18 (dd, 1H, J=8.5, 8.5 Hz), 3.75 (m, 1H), 3.47 (d, 1H, J=8.17 Hz),3.01 (d, 1H, J=3.8 Hz), 2.78 (s, 3H), 1.55 (m, 1H), 0.98 (d, 3H, J=6.61Hz), 0.83 (d, 3H, J=6.79 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ159.38, 72.33,61.92, 59.85, 30.47, 29.08, 19.40, 18.95. FABMS found 174.1 [M+H⁺].

(4R,5R)-3-N-Methyl-4-methylester-5-isopropyl-2-oxazolidinone 3.11

A solution of the oxazolidinone alcohol 3.16 (600 mg, 3.47 mmol) inacetone (16 ml) at 0° C. was treated with Jones reagent (1.55 ml,prepared by adding 5.34 g of CrO₃ dropwise to 4.6 ml conc. H₂SO₄ anddiluting with water to 20 ml) and stirred for 1 hour at roomtemperature. An additional 0.400 ml of Jones reagent was added and thereaction was stirred for another hour. After decomposing the remainingJones reagent with isopropanol, the reaction mixture was decanted intoanother flask and the remaining solids were dissolved in satd. NaCl andextracted with CHCl₃ (3×50 ml). The organic layers were combined, driedover Na₂SO₄, filtered and concentrated in vacuo to give 3.10 which wasused directly in the next reaction.

Acid 3.10 was dissolved in 3:1 benzene/MeOH (36 ml) and treated withTMS-diazomethane (3.47 ml, 2 M in hexanes). The reaction mixture wasstirred for 1 hour at room temperature and concentrated in vacuo. Theresidue was purified by flash chromatography (40% EthOAc/hexane) to give490 mgs (70% over two steps) of the methyl ester 3.11 as a colorlessoil. R_(f)=0.25 (40% EthOAc/hexane). [αI_(D) ²³=−2.7 (c 0.38, CDCl₃), ¹HNMR (300 MHz, CDCl3₃) δ4.26 (d, 1H, J=7.6 Hz), 4.19 (dd, 1H, J=9.1, 7.6Hz), 3.82 (s, 3H), 2.85 (s, 3H), 1.86-1.71 (m, 1H), 1.07 (d, 3H, J=6.5Hz), 0.98 (d, 3H, J=6.6 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ168.89, 81.04,63.81, 52.49, 42.08, 30.25, 29.3, 18.99, 18.44. HR-EI: calculated forC₉H₁₅NO₄ 201.1001, found 201.0992.

Example 2 Synthesis of CsA Analogs Boc-MeLeu-Lys(2Cl-Cbz)-OBn 4.1

Following general procedure A, the title compound was synthesized in 80%yield by coupling of the HCl salt of H-Lys(2Cl-Cbz)-OBn (8.56 g, 19.4mmol) to Boc-MeLeu-OH (5.24 g, 21.3 mmol) in DCM (130 ml) with BOP-Cl(5.43 g, 21.3 mmol) and DIEA (10.5 ml, 60.1 mmol) to give 9.78 g of thedipeptide. R_(f)=0.29 (30% EthOAc/hexane), [α]_(D) ²³=−53.01 (c 0.415,CHCl₃), FABMS (C₃₃H₄₆N₃O₇Cl) found 632.1.

Boc-Val-Melxu-Lys(2Cl-Cbz)-OBn 4.2

Following general procedure D, the Boc group inBoc-MeLeu-Lys(2Cl-Cbz)-OBn 4.1 (9.78 g, 15.5 mmol) was cleaved to formthe HCl salt of MeLeu-Lys(2Cl-Cbz)-OBn, which was coupled to Boc-Val-OH(3.7 g, 17.0 mmol) in DCM (105 ml) with BOP-Cl (4.33 g, 17.0 mmol) andDIEA (8.35 ml, 48.0 mmol) via general procedure B to give 6.68 g (59%yield) of the title compound. R_(f)=0.30 (40% EthOAc/hexane), [α]_(D)²³=−59.30 (c 0.860, CHCl₃), FABMS (C₃₈H₅₅N₄O₈Cl) found 731.2.

Boc-MeLeu-Val-MeLcu-Lys(2Cl-Cbz)-OBn (SEQ. ID. NO: 2) 4.3

Following general procedure D, the Boc group inBoc-Val-MeLeu-Lys(2Cl-Cbz)-OBn 4.2 (6.68 g, 9.13 mmol) was cleaved toform the HCl salt of Val-MLeu-Lys(2Cl-Cbz)-OBn, which was coupled toBoc-MeLeu-OH (2.463 g, 10.04 mmol) in DCM (60 ml) with BOP-Cl (2.553 g,10.04 mmol) and DIEA (4.93 ml, 28.3 mmol) via general procedure A togive 5.367 g (69% yield) of the title compound. R_(f)=0.37 (50%EthOAc/hexane), [α]_(D) ²³=−94.33 (c 0.670, CHCl₃), FABMS (C₄₅H₆₈N₅O₉Cl)found 858.3.

Boc-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2 Cl-Cbz)-OBn (SEQ. ID. NO: 3) 4.4

Following general procedure E, the Boc group inBoc-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn 4.3 (773 mg, 0.886 mmol) wascleaved to form H-Meleu-Val-MeLeu-Lys(2Cl-Cbz)-OBn, which was coupled toBoc-(D)MeSer(OBn)-OH-DCHA salt (464 mg, 0.946 mmol) in DCM (6 ml) withBOP-Cl (241 mg, 0.946 mmol) and DIEA (0.165 ml, 0.946 mmol) via generalprocedure A to give 638 mg (71% yield) of the title compound. R_(f)=0.33(50% EthOAc/hexane), [α]_(D) ²³=−48.09 (c 0.47, CHCl₃), FABMS(C₅₆H₈₁,N₆O₁₁,Cl) found 1049.3.

Boc-Abu-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn (SEQ. ID. NO: 4)4.5

Following general procedure E, the Boc group inBoc-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn 4.4 (618 mg, 0.589mmol) was cleaved to form H-(D)MeSer(OBn)-Val-MeLeu-Lys(2Cl-Cbz)-OBn,which was coupled to Boc-Abu-OH (247 mg, 1.22 mmol) in DCM (5 ml) withBOP-Cl (311 mg, 1.82 mmol) and DIEA (0.313 ml, 1.82 mmol) via generalprocedure B to give 450 mg (68% yield) of the title compound. R_(f)=0.33(50% EthOAc/hexane), [α]_(D) ²³=−66.10 (c 0.295, CHCl₃), FABMS(C₆₀H₈₈N₇O₁₂Cl) found 1134.4.

Acetonide-MeLeu(3-OH)-Abu-(D)MeSer(OBu)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn(SEQ. ID. NO: 5) 4.7

Following general procedure F, MeLeu(3-OH) 3.6 (50 mg, 0.31 mmol) inrefluxing acetone (100 ml) was protected as the N,O-acetonide 4.6 andused in the coupling reaction below.

Following general procedure E, the Boc group inBoc-Abu-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn 4.5 (330 mg, 0.29mmol) was cleaved to formH-Abu-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn, which was coupledto the protected MeLeu(3-OH) in THF (4.5 ml) with DCC (64 mg, 0.31mmol), HOBt (84 mg, 0.62 mmol) and NMM (0.034 ml, 0.31 mmol) via generalprocedure G to give 245 mg (70% yield) of the title compound. Thecompound was purified by flash chromatography with a 10-20-30%acetone/hexane gradient. R_(f)=0.38 (40% acetone/hexane), [α]_(D)²³=−40.0 (c 0.295, CHCl₃), FABMS (C₆₅H₉₇N₈O₁₂₂Cl) found 1217.5.

H-MeLeu(3-OH)-Abu-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn(SEQ.ID. NO: 6) 4.8

Following general procedure H, the acetonide was cleaved from theprotected heptapeptide 4.7 (235 mg, 0.193 mmol) with 1 N HCl (aq) (0.772ml) in MeOH (3.92 ml) to give 173 mgs (76% yield) of the title compound.R_(f)=0.11 (5% MeOH/DCM), [α]_(D) ²³=−88.6 (c 0.175, CHCl₃), FABMS(C₆₂H₉₃N₈O₁₂Cl) found 1177.5.

Fmoc-(D)Ala-MeLeu-Meleu-MeVal-Meleu(3OH)-Abu-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn(SEQ ID. NO: 7) 4.9

Following general procedure I,H-MeLeu-Abu-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)OBn 4.8 (170 mg,0.144 mmol) was coupled to Fmoc-(D)Ala-MeLeu-MeLeu-MeVal-OH (SEQ. ID.NO: 8) (147 mg, 0.216 mmol) in DCM (3 ml) with BOP (96 mg, 0.216 mmol)and NMM (0.040 ml, 0.36 mmol) to give 139 mg (53% yield) of the titlecompound. The compound was purified by flash chromatography with a10-20-30-40% acetone/hexane gradient. R_(f)=0.55 (50% acetone/hexane),[α]_(D) ²³=−95.5 (c 0.20, CHCl₃), FABMS (C₁₀₀H₁₄₅N₁₂O₁₈Cl) found 1837.9.

(MeLeu(3-OH)¹, (D)MeSer(OBn)³, Lys(2Cl-Cbz)⁷)-CsA (SEQ. ID. NO: 9) 4.10

Following general procedure J,Fmoc-(D)Ala-MeLeu-MeLeu-MeVal-MeLeu(3-OH)-Abu(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn 4.9 (133 mg, 0.0724 mmol) was treatedwith 0.2 N NaOH (0.8 ml) in ethanol (3.5 ml) to give the deprotectedundecapeptide, which was cyclized by using propylphosponic anhydride(0.054 ml, 50% (v/v) in DCM) and DMAP (49 mg, 0.398 mmol) in DCM (345ml). The compound was purified by flash chromatography (10-20-30%acetone/hexane) to give 72 mg (66% yield) of the CsA analog 4.10.R_(f)=0.46 (50% acetone/hexane), FABMS (C₇₈H₁₂₇N₁₂O₁₅Cl) found 1507.7.

Boc-(D)MeSer(OTBS)-OH 4.13

A solution of Boc-(D)MeSer-OH 4.12 (3.641 g, 16.47 mmol) in DMF (80 ml)was treated with TBS-Cl (12.413 g, 82.35 mmol) and imidazole (11.213 g,164.7 mmol). The reaction was stirred overnight at room temperature, andthen concentrated from toluene in vacuo on the high-vacuum. The residuewas suspended in H₂O, acidified to pH 4 with 10% citric acid, andextracted with ether (3×). The combined organic layers were dried overNa₂SO₄, filtered, and concentrated in vacuo. The residue was purified byflash chromatography (100% DCM, then 7-10% MeOH/DCM) to give 3.404 g(62% yield) of the title compound. R_(f)=0.68 (95:4:1 DCM/MeOH/HOAc),[α]_(D) ²³=+4.4 (c 0.635, CHCl₃), ¹H NMR (30 Q MHz, CDCl₃) (mixture ofcis-trans isomers) δ4.68-4.28 (m, 1H), 4.06-3.85 (m, 2H), 2.95-2.81 (m,3H), 1.46-1.36 (m, 9H), 0.90-0.78 (m, 9H), 0.10-(−) 0.03 (m, 6H).

Boc-(D)MeSer(OTBS)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn (SEQ. ID. NO: 10)4.15

Following general procedure E, the Boc group inBoc-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn 4.3 (7.966 g, 9.279 mmol) wascleaved to form H-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn, which was coupled toBoc-(D)MeSer(OTBS)-OH 4.13 (3.405 g, 10.21 mmol) in DCM (62 ml) withBOP-Cl (2.596 g, 10.21 mmol) and DIEA (3.395 ml, 19.49 mmol) via generalprocedure A to give 5.38 g (58% yield) of the title compound. R_(f)=0.60(60% EthOAc/hexane), [α]_(D) ²³=−48.24 (c 0.425, CHCl₃), FABMS(C₅₅H₈₈N₆O₁₁ClSi) found 1073.6.

Boc-(D)MeSer-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn (SEQ. ID. NO: 11) 4.14

A solution of Boc-(D)MeSer(OTBS)-MeLeu-Lys(2Cl-Cbz)-OBn 4.15 (5.23 g,4.87 mmol) in THF (88 ml) was treated with HF/pyridine stock solution(74 ml of a stock solution prepared from 18.75 g HF/pyridine, 18.8 mlpyridine, and 75 ml THF) and the reaction was stirred at roomtemperature for 5 hours. The reaction mixture was combined with 75 ml of1 N NaHCO₃, and extracted with DCM (3×250 ml). The organic layers werecombined, dried over Na₂SO₄, filtered, and concentrated in vacuo. Theresidue was purified by flash chromatography (60% EthOAc/hexane) to give4.15 g (89% yield) of the title compound. R_(f)=0.10 (60%EthOAc/hexane), [α]_(D) ²³=−45.0 (c 0.5, CHCl₃), FABMS (C₄₉H₇₅N₆O₁₁Cl)found 959.5.

Boc-Abu-(D)MeSer-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn (SEQ. ID. NO: 12) 4.16

Following general procedure E, the Boc group inBoc-(D)MeSer-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn 4.14 (934 mg, 0.975 mmol)was cleaved to form H-(D)MeSer-Val-MeLeu-Lys(2Cl-Cbz)-OBn, which wascoupled to Boc-Abu-OH (217 mg, 1.07 mmol) in DCM (6.5 ml) with BOP-Ci(272 mg, 1.07 mmol) and DIEA (0.357 ml, 2.05 mmol) via general procedureB to give 693 mg (68% yield) of the title compound. R_(f)=0.36 (80%EthOAc/hexane), [α]_(D) ²³=−48.4 (c 0.57 CHCl₃), FABMS (C₅₃H₈₂N₇O₁₂Cl)m/z 1044.5.

Acetonide-MeLeu(3-OH)-Abu-(D)MeSer-MeLeu-VaI-MeLeu-Lys(2Cl-Cbz)-OBn(SEQ. ID. NO; 13) 4.17

Following general procedure F, MeLeu(3-OH) 3.16 (509 mg, 3.16 mmol) inrefluxing acetone (1400 ml) was protected as the N,O-acetonide 4.6 andused in the coupling reaction below.

Following general procedure E, the Boc group inBoc-Abu-(D)MeSer-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn 4.16 (3 g, 2.87 mmol)was cleaved to formH-Abu-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn, which was coupledto the protected MeLeu(3-OH) 4.6 in THF (49 ml) with DCC (652 mg, 3.16mmol), HOBt (852 mg, 3.16 mmol) and NMM (0.347 ml, 3.16 mmol) viageneral procedure G to give 2.58 mg (80% yield) of the title compound.The compound was purified by flash chromatography with a 20-30-40-50%acetone/hexane gradient. R_(f)=0.6 (50% acetone/hexane), [α]_(D)²³=−44.1 (c 0.365, CHCl₃), FABMS (C₅₈H₉₁N₈O₁₂Cl) found 1127.5.

H-MeLeu(3-OH)-Abu-(D)MeSer-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn (SEQ. ID.NO: 14) 4.18

Following general procedure H, the acetonide was cleaved from theprotected heptapeptide 4.17 (2.55 g, 2.26 mmol) with 1 N HCl (aq) (9.04ml) in MeOH (45 ml) to give 1.65 g (67% yield) of the title compound.R_(f)=0.25 (10% MeOH/DCM), [α]_(D) ²³=−41.5 (c 0.585, CHCl₃), FABMS(C₅₅H₈₇N₈O₁₂Cl) m/z 1087.5.

Fmoc-(D)Ala-MeLeu-MeLeu-MeVal-MeLeu(3-OH)-Abu-(D)MeSer-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn(SEQ. ID. NO: 15) 4.19

Following general procedure I,H-MeLeu-Abu-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn 4M (1.63 g,1.48 mmol) was coupled to Fmoc-(D)Ala-MeLeu-MeLeu-MeVal-OH (1.13 g, 1.92mmol) in DCM (30 ml) with BOP (851 mg, 2.22 mmol) and NMM (0.325 ml,2.96 mmol) to give 1.3 g (50% yield) of the title compound. The compoundwas purified by flash chromatography with a 20-30-40% acetone/hexanegradient. R_(f)=0.46 (50% acetone/hexane), [α]_(D) ²³=−107.8 (c 0.45,CHCl₃), FABMS (C₉₃H₁₃₉N₁₂O₁₈Cl) found 1749.0.

(MeLeu(3-OH)¹, (D)MeSer(OBn)³, Lys(2Cl-Cbz)⁷)-CsA (SEQ. ID. NO: 16) 4.20

Following general procedure I,Fmoc-(D)Ala-MeLeu-MeLeu-MeVal-MeLeu(3-OH)-Abu-(D)MeSer-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn(1.25 g, 0.715 mmol) 4.19 was treated with 0.2 N NaOH (7.85 ml+3.9 ml)in ethanol (36 ml) to give the deprotected undecapeptide, which wascyclized by using propylphosponic anhydride (0.526 ml, 50% (v/v) in DCM)and DMAP (480 mg, 3.93 mmol) in DCM (345 ml). The compound was purifiedwith flash chromatography (10-20-30% acetone/hexane) to give 531 mg (53%yield) of the title compound. R_(f)=0.42 (50% acetone/hexane), FABMS(C₇₁H₁₂₁N₁₂O₁₅Cl) found 1417.8.

Boc-MeAla-Lys(2Cl-Cbz)-OBn 11.1

Following general procedure A, the title compound was synthesized in 80%yield by coupling of the HCl salt of H-Lys(2Cl-Cbz)-OBn (8.81 g, 20.8mmol) to Boc-MeAla-OH (4.65 g, 22.9 mmol) in DCM (140 ml) with BOP-Cl(5.83 g, 21.3 mmol) and DIEA (11.24 ml, 65.5 mmol) to give 9.82 g of thedipeptide. R_(f)=0.50 (50% EthOAc/hexane), [α]_(D) ²³=−38.6 (c 0.515,CHCl₃), FABMS (C₃₀H₄₀N₃O₇Cl) found 590.3.

Boc-Val-MeAla-Lys(2Cl-Cbz)-OBn 11.2

Following general procedure D, the Boc group inBoc-MeLeu-Lys(2Cl-Cbz)-OBn 11.1 (9.753 g, 16.5 mmol) was cleaved to formthe HCl salt of MeLeu-Lys(2Cl-Cbz)-OBn, which was coupled to Boc-Val-OH(3.957 g, 18.2 mmol) in DCM (110 ml) with BOP-Cl (4.63 g, 18.2 mmol) andDIEA (8.935 ml, 51.3 mmol) via general procedure B to give 7.23 g (63%yield) of the title compound. R_(f)=0.30 (60% EthOAc/hexane), [α]_(D)²³=−92.6 (c 0.685, CHCl₃), FABMS (C₃₅H₄₉N₄O₈Cl) found 689.4.

Boc-MeAla-Val-MeAla-Lys(2Cl-Cbz)-OBn (SEQ. ID. NO: 8) 11.3

Following general procedure D, the Boc group inBoc-Val-MeAla-Lys(2Cl-Cbz-OBn 11.2 (7.27 g, 10.5 mmol) was cleaved toform the HCl salt of Val-MeLeu-Lys(2Cl-Cbz)-OBn, which was coupled toBoc-MeAla-OH (2.35 g, 11.6 mmol) in DCM (70 ml) with BOP-Cl (2.94 g,11.6 mmol) and DIEA (5.7 ml, 32.6 mmol) via general procedure A to give3.50 g (43% yield) of the title compound. R_(f)=0.30 (80%EthOAc/hexane), [α]_(D) ²³=−90.6 (c 0.545, CHCl₃), FABMS (C₃₉H₅₆N₅O₉Cl)found 774.4.

Boc-Abu-Sar-MeAla-Val-MeAla-Lys(2Cl-Cbz)-OBn (SEQ. ID. NO: 17) 11.4

Following general procedure E, the Boc group inBoc-MeAla-Val-MeAla-Lys(2Cl-Cbz)-OBn 11.3 (665 mg, 0.86 mmol) wascleaved to form H-MeAla-Val-MeAla-Lys(2Cl-Cbz)-OBn, which was coupled toBoc-Abu-Sar-OH (260 mg, 0.946 mmol) in DCM (9 ml) with BOP-Cl (240 mg,0.946 mmol) and DIEA (0.314 ml, 1.81 mmol) via general procedure A togive 576 mg (72% yield) of the title compound. The compound was purifiedby flash chromatography (50% acetone/hexane). R_(f)=0.20 (50%acetone/hexane), [α]_(D) ²³=−92.7 (c 0.655, CHCl₃), FABMS(C₄₆H₆₈N₇O₁₁Cl) found 952.5 (M+Na⁺).

Acetonide-MeLeu(3-OH)-Abu-Sar-MeAla-Val-MeAla-Lys(2Cl-Cbz)-OBn (SEQ. IDNO: 18) 11.5

Following general procedure F, MeLeu(3-OH) 3.6 (240 mg, 1.49 mmol) inrefluxing acetone (240 ml) was protected as the N,O-acetonide 4.8 andused in the coupling reaction below.

Following general procedure E, the Boc group inBoc-Abu-Sar-MeAla-Val-MeAla-Lys(2Cl-Cbz)-OBn 11.4 (1.26 g, 1.36 mmol)was cleaved to form H-Abu-Sar-MeAla-Val-MeAla-Lys(2Cl-Cbz)-oBn, whichwas coupled to the protected MeLeu(3-OH) in THF (20.5 ml) with DCC (308mg, 1.49 mmol), HOBt (403 mg, 2.98 mmol) and NMM (0.164 ml, 1.49 mmol)via general procedure G to give 920 mg (67% yield) of the titlecompound. The compound was purified by flash chromatography with a30-40-50% acetone/hexane gradient. R_(f)=0.26 (50% acetone/hexane),[α]_(D) ²³=−94.8 (c 0.81, CHCl₃), FABMS (C₅₁H₇₇N₈O₁₁Cl) m/z 1013.7.

H-MeLeu(3-OH)-Abu-Sar-MeAla-Val-MeAla-Lys(2Cl-Cbz)-OBn (SEQ. ID. NO: 19)11.6

Following general procedure H, the acetonide was cleaved from theprotected heptapeptide 11.5 (862 mg, 0.85 mmol) with 1 N HCl (aq) (3.4ml) in MeOH (17 ml) to give 700 mgs (84% yield) of the title compound.The compound was purified by flash chromatography with a 4-5% MeOH/DCMgradient. R_(f)=0.33 (10% MeOH/DCM), [α]_(D) ²³=−95.6 (c 0.455, CHCl₃),FABMS (C₄₈H₇₃N₈O₁₁Cl) found 973.5.

Fmoc-(D)Ala-MeLeu-MeLeu-MeVal-MeLeu(3-OH)-Abu-(D)MeSer(OBn)-MeLeu-Val-MeLeu-Lys(2Cl-Cbz)-OBn(SEQ. ID. NO: 20) 11.7

Following general procedure I, theH-MeLeu-Abu-Sar-MeAla-Val-MeAla-Lys(2Cl-Cbz)-OBn 11.6 (670 mg, 0.69mmol) was coupled to Fmoc-(D)Ala-MeLeu-MeLeu-MeVal-OH (640 mg, 0.943mmol) in DCM (14 ml) with BOP (457 mg, 1.03 mmol) and NMM (0.189 ml,1.72 mmol) to give 658 mg (59% yield) of the title compound. Thecompound was purified by flash chromatography with a 20-30-40-50%acetone/hexane gradient. R_(f)=0.39 (50% acetone/hexane), [α]_(D)²³=−133.0 (c 0.43, CHCl₃), FABMS (C₈₆H₁₂₅N₁₂O₁₇Cl) found 1634.4.

(MeLeu(3-OH)¹, MeAla^(4.6), Lys(2Cl-Cbz)⁷)-CsA (SEQ. ID. NO: 21) 11.8

Following general procedure I,Fmoc-(D)Ala-MeLeu-MeLeu-MeVal-MeLeu(3-OH)-Abu-Sar-MeAla-Val-MeAla-Lys(2Cl-Cbz)-OBn11.7 (640 mg, 0.391 mmol) was treated with 0.2 N NaOH (4.3 ml+2.1 ml) inethanol (20 ml) to give the deprotected undecapeptide, which wascyclized by using propylphosponic anhydride (0.288 ml, 50% (v/v) in DCM)and DMAP (263 mg, 2.15 mmol) in DCM (1.86 L). The compound was purifiedwith flash chromatography (10-20-30-40-50% acetone/hexane) to give 380mg (75% yield) of the title compound. R_(f)=0.32 (50% acetone/hexane),FABMS (C₆₄H₁₀₆N₁₂O₁₄Cl) found 1303.8.

Acetylcyclosporin A 4.21

A solution of CsA (200 mg, 0.168 mmol) in acetic anhydride (3.5 ml) wastreated with DMAP (41 mg, 0.337 mmol) and the reaction was stirred for48 hours at room temperature. The reaction was poured into H₂O (25 ml)and ether (25 280 ml), phases were separated, and the aqueous phase wasextracted with ether (2×25 ml). The organic phases were combined, driedover Na₂SO₄, filtered, and concentrated in vacuo. The residue waspurified by flash chromatography (20-30-40% acetone/hexane) to give 136mgs of the title compound (65% yield). FABMS calculated forC₆₄H₁₁₃N₁₁O₁₃ 1244.6, found 1244.8.

η-Acetoxyacetylcyclosporin A 4.23

A solution of acetylcyclosporin A in CCl₄ was treated withazobisisobutyronitrile (AIBN) and N-bromosuccinimide (NBS). The reactionwas refluxed for 2.5 hours and then concentrated in vacuo. The residuewas brought up in ether and filtered through celite. The filtrate waswashed with H₂O, dried over Na₂SO₄, filtered, and concentrated in vacuoto give crude 4.22. The residue was dissolved in methyl ethyl ketone,and tetraethylammonium acetate hydrate and Nal (cat.) were added to thesolution. The reaction was stirred at 60-80° C. for 3 hours and thenroom temperature for 2 days, diluted with methyl tert-butyl ether andwashed with H₂O, and brine. The organic layer was dried over Na₂SO₄,filtered, and concentrated in vacuo. The residue was purified by flashchromatography (20-30-40-50% acetone/hexane) to give 42 mgs (47% yield)of the title compound. FABMS calculated for C₆₆H₁₁₅N₁₁O₁₅ 1301.9, found1302.

η-Hydroxycyclosporin A (OL-17) 2.11

A solution of the diacetate 4.23 in MeOH was treated with NaOMe (4 eq.)and the reaction was stirred for 2.5 hours at room temperature. Themixture was concentrated in vacuo, the residue was dissolved in methyltert-butyl ether, washed with H₂O, brine, and 1 N NaHCO₃, dried overNa₂SO₄ filtered, and concentrated in vacuo. The residue was purified byflash chromatography (40% acetone/hexane) to give 32 mgs (50% yield) ofthe title compound. FABMS calculated for C₆₂N₁₁₁N₁₁O₁₃1217.9, found1218.9.

OL17 p-Nitrophenol carbonate

A solution of η-hydroxycyclosporin A (28 mg, 0.0227 mmol) andp-nitrophenol chloroformate (0.025 mmol) in DCM (0.5 ml) was treatedwith DIEA (0.004 ml, 0.025 mmol) and the reaction was stirred overnightat room temperature. The reaction was concentrated in vacuo and theresidue was purified by flash chromatography (2-3% MeOH/DCM) to give 23mgs (74% yield) of the carbonate. FABMS found 1383.7.

Example 3 Synthesis of CS-131 Analog and CS-131 ConjugateBoc-Ile-Phe-Oallyl

According to general procedure C, Boc-Ile-OH (3.07 g, 13.3 mmol) wascoupled to the HCl salt of Phe-Oallyl (14.6 mmol) in CH₂Cl₂ (60 ml) andDMF (6 ml) by using TEA (1.94 ml, 13.9 mmol), HOBt (3.047 g, 19.9 mmol),and EDCI (2.79 g. 14.6 mmol). The product was purified by flashchromatography using 30% EthOAc/Hexane as the eluant to give 78% yieldthe title compounds an oil. R_(f)=0.38 (40% EthOAc/hexane). [α]_(D)²³=+15.6 (c 0.455, CHCl). FABMS (C₂₃H₃₄N₂O₅) found 419.2 (M+H⁺).

Boc-Pro-Ile-Phe-Oallyl 5.6

After cleavage of the Boc group from Boc-Ile-Phe-Oallyl according togeneral procedure D, the HCl salt of Ile-Phe-Oallyl was coupled toBoc-Pro in DMF (60 ml) via general procedure C, by using TEA (1.94 ml,13.9 mmol), HOBt (3.047 g, 19.9 mmol), and EDCl (2.79 g. 14.6 mmol).After workup, the product was crystallized from cold EthOAc to give thetitle compound in 73% yield as a white solid. R_(f=)0.30 (50%EthOAc/hexane). [α]_(D) ²³=−60.5 (c 0.585, CHC₃). FABMS (C₂₈H₄₁N₃O₆)found 516.3 (M+H⁺).

N-tert-Butoxycarbonyl-phenylalanine-N-methoxy-N-methylamide 5.2

To a solution of Boc-Phe-OH 5.1 (2.0 g, 7.53 mmol) and N,Odimethylhydroxylamine (808 mg, 8.29 mmol) in DCM (38 ml) was added TEA(2.2 ml, 15.8 mmol) and HOBT (1.27 g, 8.29 mmol). After cooling to 0°C., EDCI (1.59 g, 8.29 mmol) was added in one portion, and the reactionwas stirred overnight, warming to room temperature. The reaction wasconcentrated in vacuo, diluted with EthOAc (150 ml), and washed withH₂O, 1 N KHSO₄, 1 N NaHCO₃ and brine. After drying over Na₂SO₄,filtering, and concentrating in vacuo, the residue was purified by flashchromatography to give the title compound as an oil in 88% yield.R_(f)=0.35 (50% EthOAc/hexane). ¹H NMR (300 MHz, CDCl₃) δ7.28 (d, 2H,J=7.4 Hz), 7.24 (t, 1H, J=6.8 Hz), 7.17 (d, 2H, 7.1 Hz), 5.18 (d, 1H,J=7.4 Hz), 4.9 (dd, 1H, J=7.0, 11.9 Hz), 3.66 (s, 3H), 3.16 (s, 3H),3.05 (dd, 1H, J=6.0, 13.6 Hz), 2.87 (dd, 1H, J=7.0, 13.6 Hz), 1.39 (s,9H).

(S)-(3-(tert)-Butoxycarbonyl)amino)-4-phenyl-1-butene 5.4

A suspension of LAH (300 mg, 7.92 mmol) in THF (16 ml) at −40° C. wastreated dropwise with a solution of Boc-Phe-NMe(OMe) 5.2 (2.05 g, 6.6mmol) in THF (8 ml). The reaction was stirred at −25° C. for 1 hour andthen cooled to −35° C. and quenched carefully by addition of 1 N KHSO₄(8ml). The suspension was taken up in 20 ml KHSO₄ and extracted with Et₂O(3×50 ml). The organic phases were combined and washed with 10% citricacid (2×30 ml), water, 5% NaHCO₃ and brine, dried over Na₂SO₄, andconcentrated in vacuo to give crude aldehyde 5.3. The residue was driedon the vacuum pump for 2 hours over P₂O₅ and taken directly on to thenext reaction.

To a solution of TMSCH₂MgCl (29 ml, 1.0 M in Et₂O) at −78° C. was addeda solution of the crude aldehyde 5.3 in THF (8 ml) via canula over 5 mm.After the grey, cloudy mixture was stirred overnight at roomtemperature, it was poured into a slurry of ice (25 g) and 10% citricacid (25 ml), and then extracted with Et₂O (3×50 ml). The organic phaseswere combined and washed with brine, dried over Na₂SO₄, filtered andconcentrated in vacuo. After drying on the vacuum pump for 2 hours, theoil was taken up in DCM (16 ml) and cooled to 0° C. BF₃OEt₂ (3.6 ml,29.3 mmol) was added dropwise over 5 min and the reaction mixture wasstirred at room temperature for 5 days. The reaction was cooled to 0°C., quenched with 2.5 N NaOH (50 ml), and extracted with 3×DCM. Theorganic layers were combined, dried over Na₂SO₄, filtered, andconcentrated in vacuo. The resulting brown oil was taken up in DCM (14ml), treated with di-tert-butyl carbonate (1.18 g, 5.43 mmol) and TEA(0.076 ml, 0.543 mmol), and stirred overnight at room temperature. Afterwashing the mixture with 10% citric acid, water, and 1 N NaHCO₃, it wasdried over Na₂SO₄ and concentrated in vacuo. The residue was purified byflash chromatography using 4:1 hexanes/EthOAc as the eluant to give 900mgs (57 % yield) of a white solid. R_(f)=0.20 (10% EthOAc/hexane). ¹HNMR (300 MHz, CDCl₃) δ7.35-7.15 (m, SH), 5.8 (m, 1H), 5.12 (d, 1H, J=10Hz), 5.07 (d, 1H, J=2.8 Hz), 4.40 (br s, 2H), 2.85 (d, 1H), 1.41 (s, 9H,J=5.8 Hz).

1-(R,S)-(1′(S)-(tert-Butoxycarbonylamino)-2-phenylethyl)oxirane 5.5

mCPBA (2.52 g, 14.6 mmol) was added to a solution of Boc-Phe-alkene 5.4(900 mg, 3.64 mmol) in DCM (36 ml) and the mixture was refluxed for 20hours. The reaction was diluted with ether and washed with sat. Na₂SO₃(2×), 1 N NaHCO₃ (3×), H₂O, and brine, dried over Na₂SO₄, filtered, andconcentrated in vacuo. The residue was purified by flash chromatographyusing 85:15:1 hexane/MTBE/isopropanol as the eluant to give 528 mgs (55%yield) of 5.5 as a white solid. R_(f)=0.18 (85:15:1hexane/MTBE/isopropanol). ¹H NMR (300 MHz, CDCl₃) δ7.37-7.18 (m, SH),4.50 (br s, 1H), 4.13 (br s, 1H), 3.05-2.82 (m, 5H), 2.70 (t, 1H, J=4.2Hz), 2.59 (br s, 1H), 1.39 (5, 9H).

Boc-Phe-(HEA)-Pro-Ile-Phe-Oallyl (SEQ. ID. NO: 22) 5.8

After cleavage of the Boc group according to general procedure D, theHCl salt of Pro-Ile-Phe-OAllyl (412 mg, 0.911 mmol) was added to asolution of Boc-Phe-epoxide 5.5 (200 mg, 0.759 mmol) and TEA (0.128 ml,0.911 mmol) in MeOH (8 ml) and the mixture was refluxed overnight. Thereaction was concentrated in vacuo and the residue was purified by flashchromatography using a 1-3% MeOH/DCM gradient as the eluant to give 396mgs (80% yield) of 5.8 as a white solid. R_(f)=0.32 (60% EthOAc/hexane).[α]_(D) ²³=−27.6 (c 0.66, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ8.3-8.2 (m,1H), 7.33-7.12 (m, 11H), 7.06-7.00 (m, 1H), 5.94-5.78 (m, 1H), 5.35-5.15(m, 3H), 4.97-4.87 (m, 1H), 4.61 (d, 2H, J=5.8 Hz), 4.39-4.27 (m, 2H),3.82-3.6 (m, 2H), 3.22-3.07 (m, 4H), 2.99-2.82 (m, 2H), 2.76-2.64 (m,1H), 2.53-2.41 (m. 1H). 2.34-2.09 (m, 2H), 1.91-1.64 (m, 4H), 1.36 (s,9H), 1.18-1.01 (m, 1H), 0.96-0.80 (m, 6H). FABMS (C₃₈H₅₄N₄O₇) found679.4 (M+H⁺).

Cbz-Asn(Trt)-OH 5.10

Concentrated H₂SO₄ (0.2 ml) was added to a solution of Cbz-Asn-OH (10 g,37.6 mmol), trityl alcohol (20 g, 75.2 mmol), and acetic anhydride (7.1ml, 75.2 mmol) in acetic acid (114 ml) and the reaction was stirred for1 hour at 50° C. The solution was cooled and slowly added to 1.0 L ofcold H₂O. The white precipitate was filtered, dissolved in EthOAc (250ml), washed with H₂O, dried over Na₂SO₄ and filtered. The resultingwhite solid was crystallized from EthOAc/hexane to give 13 g (68%) of awhite solid.

Cbz-Asn(Trt)-Phe-(HEA)-Pro-Ile-Phe-Oallyl (SEQ. ID. NO: 23) 5.11

After cleavage of the Boc group according to general procedure D, theUses resulting HCl salt of Phe-(HEA)-Pro-Ile-Phe-Oallyl (188 mg, 0.306mmol) was added to a solution of Cbz-Asn(Trt)-OH 5.10 (202 mg, 0.397mmol), DIEA (0.069 ml, 0.397 mmol), and HOBt (70 mg, 0.52 mmol) in DMF(3 ml) and the reaction was cooled to 0° C. EDCI was added in oneportion and the reaction was stirred overnight, diluted with EthOAc (50ml), and washed with H₂O, KHSO₄, NaHCO₃, and brine. The organic phasewas dried over Na₂SO₄, filtered and concentrated in vacuo. The residuewas purified by flash chromatography using a 1-4% MeOH/DCM gradient asthe eluant to give 225 mgs (68% yield) of 5.11 as a white solid.R_(f)=0.60 (90:10 DCM:MeOH). [α]_(D) ²³=−26.3 (c 0.40, CHCl₃). ¹H NMR(300 MHz, CDCl₃) δ8.09-8.01 (m, 1H), 7.35-7.08 (m, 30H), 6.93-6.88 (m,1H), 6.62-6.49 (m, 1H), 6.31-6.2 (m, 1H), 5.92-5.73 (m, 1H), 5.31-5.18(m, 2H), 5.14-4.98 (m, 2H), 4.94-4.85 (m, 1H), 4.6-4.56 (m, 2H),4.48-4.36 (m, 1H), 4.28-4.17 (m, 1H), 4.06-3.94 (m. 1H). 3.73-3.61 (m,1H), 3.19-3.03 (m, 4H), 2.97-2.79 (m, 2H), 2.76-2.42 (m, 4H), 2.35-2.21(m, 1H), 2.18-1.98 (m, 2H), 1.89-1.56 (m, 4H), 1.45-1.31 (m, 1H),1.04-0.97 (m, 1H), 0.93-0.80 (m, 6H). FABMS (CHNO) found 1069.5 (M+H⁺).

CS-131-(MeLeu(3-OH)¹, (D)MeSer³, Lys⁷)CsA Conjugate 2.8

A solution of Cbz-Asn(Trt)-Phe-(HEA)-Pro-Ile-Phe-Oallyl 5.11 in THF/MeOH(1:1) was treated with LIOH (aq) and the reaction was stirred for 3hours at room temperature. After the reaction was concentrated in vacuo.the residue was dissolved in H₂O, acidified with 1 N HCl, and extractedwith DCM. The combined organic layers were dried over Na₂SO₄, filtered,and concentrated in vacuo to give crude acid 5.12.

A solution of the trityl-protected acid 5.12 in DCM (2 ml) was treatedwith TFA (0.5 ml) and the reaction was stirred 1 hour at roomtemperature and then concentrated in vacuo. The residue was concentratedseveral times from ether, suspended in ether, and filtered to remove thetritylmethane and trityl alcohol. Concentration of the filtrate gave thefully deprotected inhibitor 5.13 which was used in the next reactionwithout any further purification.

A solution of the fully deprotected inhibitor 5.13 (42 mg, 0.047 mmol)and the free amine CsA analog 4.11 (58 mg, 0.047 mmol, produced from4.20 via general procedure K) in DMF/acetonitrile (1:1, 1 ml) wastreated with PyAOP (27 mg, 0.052 mmol) and DIEA (0.017 ml, 0.099 mmol),stirred overnight at room temperature, and concentrated in vacuo. Theresidue was diluted with EthOAc, washed with brine (with a few drops of1 N NaHCO₃ added), dried over Na₂SO₄, and concentrated in vacuo, to anoil which was purified by flash chromatography (2-4-6% MeOH/DCM) to give45 mg (47% yield) of conjugate 2˜. R_(f)=0.45 (9:1 DCM/MeOH). FABMSfound 2019.1 [M+H⁺].

Example 4 Synthesis of Pyrone Inhibitors and Pyrone Conjugates1-(TBS)-3-(tert)-Butyl-hydroquinone 6.2

To a solution of t-butyl hydroquinone (3.0 g, 18 mmol) in DCM (60 ml)was added TBS-Cl (2.71 g, 18 mmol) and imidazole (1.22 g, 18 mmol). Thereaction was stirred for 2 hours at room temperature, washed with KHSO₄and brine, dried over Na₂SO₄, filtered, and concentrated in vacuo. Theresidue was purified by flash chromatography (10% Et₂O/hexane) to give4.9 g (97% yield) of an off-white solid. R_(f) 0.34 (20% Et₂O/hexane).¹H NMR (300 MHz, CDCl₃) δ6.59 (d, 1H, J=2.3 Hz), 6.37-6.33 (m, 2H), 4.33(s, 1H), 1.21 (s, 9H), 0.81 (s, 9H), 0.00 (s, 6H). ¹³C NMR (75.5 MHz,CDCl₃) δ149.0, 148.4, 137.1, 118.9, 117.4, 116.9, 34.5, 29.5, 25.8,18.2, −4.5. HR-EI: calculated for C₁₆H₂₈O₂Si 280.1858, found 280.1860.

1-(TBS)-3-(tert)-butyl-4-N,N-(dimethyl)thiocarbamoylhydroquinone 6.3

A solution of 1-(TBS)-3-tert-butyl-hydroquinone 6.2(2.93 g, 10.4 mmol)in THF (75 ml) was treated with NaH (479 mgs, 12.5 mmol of a 60%dispersion in mineral oil) and the mixture stirred for 1 hour at roomtemperature. N,N dimethylthiocarbamoyl chloride (1.55 g, 12.5 mmol) wasthen added in one portion to the solution and the resulting mixture wasstirred overnight at room temperature. After quenching the reaction with1 N KHSO₄, it was diluted with EthOAc (150 ml), washed with brine,KHSO₄, NaHCO₃, brine, dried over Na₂SO₄, filtered and concentrated invacuo. The residue was purified by flash chromatography (10%Et₂O/hexane) to give 2.03 g (52% yield) of an off-white solid. R_(f)0.32 (20% Et₂O/hexane). ¹H NMR (300 MHz, CDCl₃) δ6.84 (d, 1H, J=8.7 Hz),6.82 (d, 1H, J=2.8 Hz), 6.65 (dd, 1H, J=2.9, 8.7 Hz), 3.49 (s, 1H), 3.36(s, 1H), 1.32 (s, 9H), 0.97 (s, 9H), 0.20 (s, 6H). ¹³C NMR (75.5 MHz,CDCl₃) δ188.3, 152.9, 146.5, 142.1, 125.9, 118.3, 117.2,43.4, 38.8,34.5, 30.6, 25.7, 18.2, −4.4. HR-EI: calculated for C₁₉H₃₃NO₂SiS367.2001, found 367.2008.

1-(TBS)-3-ter-Butyl-4N,N-dimethycarbamoylthiohydroquinone 6.4

A sand bath was heated to 275-300° C. and a 2.5 ml flask containing neat1-(TBS)-3-tert-butyl-4N,N-dimethylthiocarbamoylhydroquinone 6.3 (1.98 g,5.39 mmol) and fitted with a vigereaux column was immersed in the sandfor 20 minutes. After allowing the brown/black solution to cool to roomtemperature, it was purified. by flash chromatography (10-20%Et₂O/hexane gradient of 5%) to yield 1.36 g (69% yield) of 6.4 as ayellow oil. R_(f) 0.22 (20% Et₂O/hexane). ¹H NMR (300 MHz, CDCl₃)δ7.36-7.25 (m, 1H), 6.92 (d, 1H J=2.6 Hz), 6.68 (dd, 1H J=2.6, 8.4 Hz),3.07 (bs, 6H), 1.43 (m, due to multiple conformers, 9H), 0.98 (m, due tomultiple conformers, 9H), 0.21 (m, due to multiple conformers, 6H). ¹³CNMR (75.5 MHz, CDCl₃) δ167.8, 156.7, 154.6, 142.6, 118.9, 118.7, 117.7,37.0, 36.3, 30.8, 25.7, 18.2, −4.3. HR-El: calculated for C₁₉H₃₃NO₂SiS367.2001, found 367.2009.

3-tert-Butyl-4-N,N-dimethycarbamoylthiohydroquinone 6.9

A solution of1-(TBS)-3-tert-butyl-4-N,N-dimethycarbamoylthiohydroquinone (1.36 g,3.71 mmol) 6.4 in THF (24 ml) was treated with TBAF (4.44 ml, 1 N THF)and the reaction was stirred for 0.5 hours. After diluting the solutionwith EthOAc (50 ml), it was washed with KHSO₄ and brine, dried overNa₂SO₄, filtered, and concentrated in vacuo. The residue was purified byflash chromatography (30-50% EthOAc/hexane, 10% increments) to give 861mgs (92% yield) of an off-white solid. R_(f) 0.13 (20% Acetone/hexane).¹H NMR (300 MHz, CDCl₃) δ7.48 (bs, 1H), 7.15-7.08 (m, 1H), 6.81 (d, 1H,J=2.7 Hz), 6.29 (dd, 1H, J=2.7, 8.3 Hz), 3.25-2.95 (m, 6H), 1.41 (s,9H). ¹³C NMR (75.5 MHz, CDCl₃) δ170.1, 158.0, 154.5, 142.3, 115.6,115.3, 114.5, 37.2, 36.3, 30.8. HR-El: calculated for C₁₃H₁₉NO₂S253.1136, found 253.1132.

1-Allyl-3-tert-butyl-4-N,N-dimethycarbamoylthiohydroquinone 6.13

Method 1: 3-(Tert)-butyl-4-N,N-diinethylcarbainoylthiohydroquinone6.9(861 mg, 3.41 mmol) was dissolved in THF (34 ml) and the solution wascooled to 0° C. NaH was added in portions (196 mgs, 5.11 mmol, 60%dispersion in mineral oil) and the resulting slurry was stirred for 10min at room temperature. The solution was treated with allyl iodide(0.624 ml, 6.82 mmol), stirred 48 hours, diluted with EthOAc (75 ml) andwashed with brine, KHSO₄, dried over Na₂SO₄, and concentrated in vacuo.The residue was purified by flash chromatography (20% EthOAc/hexane) togive 843 mgs (85% yield) of an oil.

Method 2: 3-tert-butyl-4-N,N-dimethycarbamoylthiohydroquinone 6.9 (370mg, 1.47 mmol), K₂CO₃ (812 mg, 5.87 mmol), and allyl bromide (0.133 ml,1.62 mmol) were dissolved in acetone (7.5 ml) and refluxed for 10 hours.After cooling the reaction to room temperature, it was diluted withEthOAc (25 ml), washed with brine, dried over Na₂SO₄, and concentratedin vacuo. The residue was purified by flash chromatography (20%EthOAc/hexane) to give 352 mgs (82% yield) of an oil. R_(f) 0.19 (20%EtOAc/hexane). ¹H NMR (300 MHz, CDCl₃) δ7.36 (d, 1H, J=8.5 Hz), 7.05 (d,1H, J=2.8 Hz), 6.78 (dd, 1H, J=2.8, 8.5 Hz), 6.14-5.98 (m, 1H),5.47-5.37 (m, 1H), 5.34-5.25 (m, 1H), 4.58-4.51 (m, 2H), 3.07 (bs, 6H),1.45 (s, 9H). ¹³C NMR C75.5 MHz, CDCl₃) δ167.9, 159.5, 154.7, 142.7,133.2, 118.4, 117.9, 115.0, 111.4,68.8, 37.0, 36.5, 30.8. HR-El:calculated for C₁₆H₂₃NO₂S 293.1449, found 293.1440.

1-(Thiotoluenesulfonate)-2-tert-butyl-4(hydroxy(3-propene))-benzene 6,14

A solution of 1-allyl-3-tert-butyl-4N,N-dimethylcarbamoylthiohydroquinone (843 mgs, 2.88 mmol) 6.13 in THF (20ml) was cooled to 0° C. and treated with LiAlH₄ (6.06 ml, 1.0 N THF)dropwise. The reaction mixture was allowed to warm room temperature over5 hours, cooled back to 0° C., and quenched with KHSO₄ (50 ml) verycarefully. The mixture was extracted with Et₂O, the phases wereseparated, and the organic layer was washed with brine, dried overNa₂SO₄, filtered, and concentrated in vacuo to give a free thiol whichwas used crude in the next reaction.

To a solution of toluenesufonyl bromide (813 mgs, 3.46 mmol) and TEACO.480 ml, 3.46 mmol) in CCl₄ (22 ml) cooled to 0° C., was addeddropwise a solution of the crude thiol in CCl₄ (7.2 ml). After stirringfor 5 min, the reaction was diluted with DCM (60 ml), washed with KHSO₄,NaHCO₃ brine, dried over Na₂SO₄, filtered and concentrated in vacuo. Theresidue was purified by flash chromatography (10% Et₂O/hexane) to give940 mgs (87% yield) of the thiosulfonate 6.14. R_(f) 0.32 (15%EtOAc/hexane). ¹H NMR (300 MHz, CDCl₃) δ7.56 (d, 1H, J=8.6 Hz), 7.46 (d,2H, J=8.4 Hz), 7.21 (d, 2H, J=8.0 Hz), 6.99 (d, 1H, J=2.8 Hz), 6.45 (dd,1H, J=2.8, 8.6 Hz), 6.15-6.09 (m, 1H), 5.45 (dd, 1H, J=17.3, 1.5 Hz),5.33 (dd, 1H, J=10.48, 1.5 Hz), 4.60-4.53 (m, 2H), 2.41 (s, 3H), 1.18(s, 9H). ¹³C NMR (75.5 MHz, CDCl₃) δ166.7, 160.6, 155.3, 144.6, 141.4,140.8, 132.7, 129.5, 127.7, 118.3, 117.8, 115.6, 111.2, 68.9, 36.7,31.0, 21.6. HR-El: calculated for C₂₀H₂₄O₃S₂ 376.1167, found 376.1157.

4-Hydroxy-pyran-2-one 6.6

A solution of phenyltrimethylsilylenol ether (5 ml, 26 mmol) in Et₂O (16ml) at −20° C. was treated with a solution of malonyl dichloride (0.842ml, 8.66 mmol) in Et₂O (4 ml) via canula. After allowing the reaction towarm to room temperature overnight, it was diluted with Et₂O (50 ml) andextracted with Na₂CO₃ (3×50 ml). The combined aqueous phases were thenwashed with Et₂O (3×50 ml), acidified with conc. HCl, and extracted withEt₂O to give 1.06 g (66% yield) of an orange solid. HR-El calculated forC₁₁H₈O₃ 188.0473, found 188.0477.

Allyl-protected Pyrone HIV Protease Inhibitor 6.15

A solution of the pyrone 6.6 (600 mgs, 3.19 mmol) and NaOH (3.2 ml, 1 Naqueous) in ethanol (18 ml) was heated in a flask fitted with a watercondensor until the pyrone was completely dissolved. To the warmreaction mixture was added dropwise a solution of the thiosulfonate 6.14(940 mgs, 2.5 mmol) in ethanol (7 ml). After refluxing the reactionmixture overnight, it was cooled to room temperature and concentrated invacuo. The residue was acidified with 3N HCl and extracted with DCM(3×50 ml). The organic layers were combined, dried over Na₂SO₄ filtered,concentrated in vacuo, and purified by flash chromatography (80:20hexane/EthOAc, then 30:65:15 hexane/EthOAc/DCM, and 50:45:15hexane/EthOAc/DCM) to give 455 mgs (45% yield) of 6.15 as a yellow foam.R_(f=)0.53 C65:30:5 DCM/EthOAc/Hexane) ¹H NMR (300 MHz, CDCl₃)δ7.91-7.68 (m, 3H), 7.55-7.42 (m, 3H), 7.01 (d, 1H, J=2.8 Hz), 6.95 (d,1H, J=8.7 Hz), 6.71 (s, 1H), 6.61 (dd, 1H, J=8.7, 2.8), 6.02 (m, 1H),5.38 (dd, 1H, J=17.3, 1.5 Hz), 5.27 (dd, 1H, J=10.5, 1.35 Hz), 4.48 (dt,2H, J=13.5, 5.4 Hz), 1.60 (s, 9H). HR-El: calculated for C₂₄H₂₄O₄S408.1395, found 408.1429.

Tert-Butyl Acetoxy Pyrone HIV Protease Inhibitor 2.4

A solution of the allyl-protected pyrone HIV protease inhibitor 6.15(132 mgs, 0.324 mmol), acetic acid (0.021 ml, 0.357 mmol), and Pd(PP₃)₄(37 mgs, 0.0032 mmol) in DCM (1.9 ml) was treated with SnBu₃H (0.96 ml,0.357 mmol) quickly in one portion. The reaction was stirred for 15minutes, changing from yellow to green to green-black, at which pointTLC showed consumption of starting material. The reaction mixture wasdiluted with DCM (10 ml) and extracted with 10% Na₂CO₃ (3×20 ml). Theaqueous layers were combined, washed with Et₂O (2×), acidified to pH 2with conc. HCl, and extracted with DCM (3×). The organic layers werecombined, dried over Na₂SO₄, and concentrated in vacuo to give 92 mgs(77% yield) of 6.16 as a yellow solid 95% clean by TLC, which was takendirectly on to the next reaction.

To a solution of pyrone hydroxyl 6.16 (50 mg, 0.136 mmol) in THF (1 ml)cooled to 0° C. was added tBuOK (32 mg, 0.285 mmol). After stirring for5 minutes, t-butylchloroacetate (0.041 ml, 0.285 mmol) andtetrabutylammonium iodide (cat.) were added and the reaction was stirredfor 5 hours warming to room temperature. The reaction was concentratedin vacuo, brought up in H₂O (5 ml), acidified to pH 3 with 1 N KHSO₄,and extracted with DCM (5×15 ml). The organic layers were combined,dried over Na₂SO₄, filtered, concentrated in vacuo, and purified byflash chromatography (40:55:5 EthOAC/hexane/DCM) to give 30 mgs (45%yield) of 2.4 as a yellow foam. R_(f)=0.34 (65:30:5 DCM/EthOAc/hexane).¹H NMR (300 MHz, CDCl₃) δ7.92-7.68 (m, 3H), 7.54-7.43 (m, 3H), 7.03 (d,1H, J=2.8 Hz), 6.95 (d, 1H, J=8.7 Hz), 6.72 (s, 1H), 6.56 (dd, 1H,J=8.7, 2.8 Hz), 4.45 (s, 2H), 1.59 (s, 9H), 1.48 (s, 9H). FABMS: found482.2 (M+H⁺).

Acetoxy Acid HIV Protease Inhibitor 6.23

A THF (1 ml) solution of the tert-butyl acetoxy pyrone inhibitor 2.4 (26mg, 0.0539 mmol) was treated with LiOH (0.162 ml, 1 N LiOH) and stirredover night at room temperature. An additional equivalent of LiOH wasadded and the solution was stirred another hour. The reaction wasconcentrated in vacuo, diluted with water, acidified with 1 N KHSO₄, andextracted with DCM and EthOAc. The combined organic layers were driedover Na₂SO₄, filtered, and concentrated in vacuo to give 22 mg (99%yield) of the acid 6.23 R_(f)=0.29 C95:4:1 DCM/MeOH/HOAc). ¹H NMR (300MHz, CDCl₃) δ7.82-7.70 (m, 2H), 7.48-7.35 (m, 3H), 6.95 (d, 1H, J=2.8Hz), 6.90 (d, 1H, J=8.7 Hz), 6.63 (s, 1H), 6.49 (dd, 1H, J=8.7, 2.8 Hz),4.45 (s, 2H), 3.85 (bs, 1H), 1.50 (s, 9H).

Boc-aminohexyl Acetoxy Amide Pyrone 6.24

The pyrone acid 6.23 (10 mg, 0.0234 mmol) and tert-butylN-(aminohexyl)-carbamate hydrochloride (6.5 mg, 0.0258 mmol) in THF (0.5ml) were treated sequentially with BOP (11 mg, 0.0258 mmol), NMM (0.011ml, 0.0725 mmol). and then stirred overnight at room temperature. Thereaction was diluted with EthOAc, washed with 10% KHSO₄ and brine, driedover Na₂SO₄, filtered, and concentrated in vacuo. The residue waspurified by flash chromatography (1% MeOH/DCM) to give 10 mg (68% yield)of the tide compound. R_(f)=0.48 (95:5 DCM/MeOH). ¹H NMR (300 MHz,CDCl₃) δ7.85-7.74 (m, 2H), 7.48-7.32 (m, 3H), 6.96 (d, 1H, J=2.8 Hz),6.89 (d, 1H, J=8.7 Hz), 6.67 (s, 1H), 6.52 (dd, 1H, J=8.7,2.8 Hz), 4.35(s, 2H), 3.26 (t, 1H, J=6.6 Hz), 3.24 (t, 1H, J=6.6 Hz), 3.08-2.97 (m,2H), 1.6-1.15 (m, 8H), 1.53 (s, 9H), 1.37 (s, 9H).

Pyrone HIV Protease Inhibitor-(MeLeu(3-OH)¹, DMeSer³, Lys⁷)-CsAConjugate 2.9

A solution of the pyrone acid 6.23 (20 mg, 0.0469 mmol) and the CsAanalog free amine 4.11 (53 mg, 0.0427 mmol, produced from 4.20 viageneral procedure K) in THF (1 ml) were treated sequentially with PyAOP(24 mg, 0.0469 mmol), NMM (0.16 ml, 0.09 mmol), and then stirredovernight at room temperature. The reaction was diluted with EthOAc,washed with 10% KHSO₄, brine, dried over Na₂SO₄, filtered, andconcentrated in vacuo. The residue was purified by flash chromatography(2-4-6% MeOH/DCM) to give 40 mg (56% yield) of conjugate 2.9. R_(f)=0.48(90:10 DCM/MeOH). FABMS found 1657.7 (M+H⁺).

Aminohexyl acetoxy amide pyrone-OL-17 semi-synthetic conjugate 2.12

A solution of aminohexyl acetoxy amide pyrone HCl salt (produced from6.24 by general procedure D) (15 mg, 0.024 mmol), TEA (0.004 ml, 0.0288mmol) and OL-17 p-nitrophenol carbonate 8.3 (15 mg, 0.0108 mmol, see CsAanalog synthetic procedures) in DMF (0.5 ml) were stirred overnight atroom temperature. The reaction was concentrated in vacuo and purified byflash chromatography (2-3-5% MeOH/DCM) to give 5 mg (28% yield) ofconjugate 2.12. R_(f)=0.55 (90:10 DCM/MeOH). FABMS found 1768.8 (M+H⁺).

Example 5 Synthesis of VX-478 Analogs and Conjugates(3S,4S)-N-Boc-4-amino-3-hydroxy-5-(4-benzyloxyphenyl)-1-pentene 7.15

A solution of Boc-Tyr(OBn)-OH 7.13 (5g, 13.5 mmol) in DMF (27 ml) wastreated with Cs₂CO₃ (8.8 g, 27 mmol) and iodomethane (0.840 ml, 13.5mmol) and the mixture was overnight at room temperature. The reactionwas then diluted with EthOAc (100 ml), and washed with H₂O, 1 N NaHCO₃,and brine. The organic phase was dried over Na₂SO₄, filtered,concentrated in vacuo to give methyl ester 7.14, which was dried overP₂O₅ on high-vacuum overnight.

Methyl ester 7.14 was dissolved in toluene (dried over sieves) andcooled to −78° C. and DIBAL (18.5 ml, 1 M in toluene) was added dropwiseover 40 minutes. After stirring for 5 minutes at −78° C.,vinylmagnesiumn bromide (71.2 ml, 1 M in THF) was added and the reactionmixture was stirred at 0° C. overnight. The reaction was cautiouslyquenched with methanol, then treated with aqueous Rochelle salts,stirred for a few minutes, and filtered. The filtrate was washed withbrine, dried over Na₂SO₄, and concentrated in vacuo. The residue waspurified by flash chromatography (10-15-20-30% EthOAc/hexane) to give2.36 g (47% yield) of the (3S,4S) allylic alcohol 7.15 and 664 mg (13%yield) of the (3R,4S) allylic alcohol 7.16.

Data for (3S,4S) diastereomer 7.15: R_(f)=0.35 (40% EtOAc/hexane).[α]_(D) ²³=−43.8 (c 0.455, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ7.48-7.27(m, SH), 7.15 (d, 2H, J=8.6 Hz), 6.93 (d, 2H, J=8.6 Hz), 5.88 (m, 1H),5.28 (m, 1H), 5.18 (m, 1H), 5.03 (s, 2H), 4.78 (m, 1H), 4.11 (bs, 1H),3.76 (m, 1H), 2.94-2.75 (m, 2H), 2.28 (bs, 1H), 1.39 (s, 9H). ¹³C NMR:CDCl₃) δ157.5, 138.4, 137.1, 130.6, 130.3, 128.6, 127.9, 127.5, 116.1,114.9, 79.5, 72.7, 70.0, 37.1, 28.3. FABMS (C₂₃H₂₉NO₄) found 384.2(M+H˜). Data for (3R,4S) diastereomer 7.16: R_(f) 0.32 (40%EtOAc/hexane). [α]_(D) ²³=−17.09 (c 0.515, CHCl₃). ¹H NMR (300 MHz,CDCl₃) δ7.46-7.28 (m, 5H), 7.10 (d, 2H, J=8.7 Hz), 6.91 (d, 2H, J=8.7Hz), 5.93 (m, 1H), 5.36 (m, 1H), 5.27 (m, 1H), 5.04 (s, 2H), 4.56 (m,1H), 4.21 (bs,. 1H), 3.93 (bs, 1H), 3.03 (bs, 1H), 2.83-2.60 (m, 2H),1.37 (s, 9H) ¹³C NMR: CDCl₃) δ157.5, 137.1, 136.9, 130.2, 128.6, 127.9,127.5, 117.0, 114.9, 79.8, 74.7, 70.0, 56.6, 35.2, 28.3. FABMSC₂₃H₂₉NO₄) found 384.2 (M+H⁺).

(2S,3S)-N-Boc-3-amino-1,2-epoxy-4-(4-hydroxypbenyl)butane 7.19

A solution of 7.15 (3.05 g, 8.07 mmol) and TEA (4.5 ml, 31.8 mmol) inDCM (66 ml) was cooled in an acetone/ice bath under argon and treateddropwise with methanesulfonyl chloride (1.24 ml, 16 mmol). The reactionwas stirred 15 minutes, quenched with 10% citric acid (75 ml), andextracted with ether (2×100 ml). The organic layers were combined,washed with H₂O and brine, dried over Na₂SO₄, and concentrated in vacuoto give an 3.55 g of 7.17 as an off-white solid which was used crude inthe next reaction. R_(f) 0.10 (40% EtOAc/hexane). [α]_(D) ²³=−41.4 (c0.500, CHCl₃). 1H NMR (300 MHz, CDCl₃) δ7.45-7.28 (m, SH), 7.16 (d, 2H,J=7.7 Hz), 6.92 (d, 2H, J=8.6 Hz), 5.93 (m, 1H), 5.46 (d, 1H, J=12.9Hz), 5.40 (d, 1H, J=7.8 Hz), 5.04 (s, 2H), 4.68 (d, 1H, J=9.3 Hz), 4.03(bs, 1H), 3.05 (s, 3H), 2.88 (dd, 1H, J=13.8, 6.4 Hz), 2.75 (dd, 1H,J=13.8, 7.0 Hz), 1.38 (s, 9H).

A solution of the crude mesylate 7.17 (3.55 g) was dissolved in DCM:MeOH(60 ml:10 ml) and cooled to −78° C. Ozone was bubbled through thesolution until a blue color persisted (ca. I hr). The reaction waspurged with argon and NaBH₄ (1.02 g, 27 mmol) was added. After stirringthe reaction at room temperature for 3 hr, it was quenched dropwise withI N HCl. The mixture was diluted with ether, washed with I N NaHCO₃,H₂O, and brine, dried over Na₂SO₄, filtered, and concentrated in vacuoto give alcohol 7.18 as a white solid (2.56 g, 70%) which was ofsufficient purity to use in the next reaction after drying. ¹H NMR (300MHz, CDCl₃) δ7.47-7.28 (m, 5H), 7.15 (d, 2H, J=8.5 Hz), 6.92 (d, 2H,J=8.6 Hz), 5.03 (s, 2H), 4.73 (t, 1H, J=6.53 Hz), 4.66 (d, 1H, J=9.5Hz), 4.21 (m, 1H), 3.82 (dd, 1H, J=12, 6.5 Hz), 3.65 (dd, 1H, J=12, 7Hz), 3.37 (m, 1H), 3.09 (s, 3H), 2.94-2.73 (m, 2H), 1.38 (s, 9H).

The alcohol mesylate 7.18 (2.55 g, 5.46 mmol) was dissolved in THF (55ml) and treated with NaH (0.209 mg, 5.46 mmol, 60% dispersion in mineraloil) in portions. The reaction was stirred at room temperature for 1 hr,refluxed for 1 hr, and then cooled to 0° C. and quenched carefully withNH₄Cl. After diluting the mixture with ether, it was washed with 1 NNaHCO₃ and brine, dried over Na2SO₄, filtered, and concentrated invacuo. The residue was purified by flash chromatography (20-30%EthOAc/hexane) to give 1.74 g (86% yield) of 7.19 as a white solid.R_(f)0.4 (40% EtOAc/hexane). [α]_(D) ²³ =+7.9 (c 0.57, CHCl₃). ¹H NMR(300 MHz, CDCl₃) δ7.46-7.29 (m, 5H), 7.14 (d, 2H, J=8.6), 6.93 (d, 2H,J=8.6), 5.05 (s, 2H), 4.41 (bs, 1H), 3.63 (bs, 1H), 2.96-2.71 (m, 5H),1.39 (s, 9H). ¹³C NMR: (CDCl₃) δ155.3, 130.5, 128.6, 128.0, 127.5,114.9, 70.0, 53.2, 46.9, 36.7, 28.3. FABMS (C₂₂H₂₇NO₄) found 370.2(M+H⁺).

(2S,3S)-N-Boc-3-amino-2-hydroxy-4-(4-hydroxyphenyl)-1-isobutylaminobutane7.20

A solution of the epoxide (800 mg, 2.15 mmol) and isobutylamine (4.27ml, 43 mmol) in methanol (11 ml) were stirred at RT for 4 hours. Afterconcentrating the residue from ether (4×) and DCM (4×) until TLC showedthe complete disappearance of the isobutylamine, the white foam wasdried on high vacuum for 2 hours and used directly in the next reactionwithout further purification.

The benzyl-protected hydroxyethylamine was dissolved in methanol (11 ml)and hydrogenated (1 atm) over Pd(OH)₂ (80 mg) for 2 hours, at which timeTLC showed the disappearance of starting material. After filteringthrough celite and concentrating in vacuo. the residue was purified byflash chromatography (small plug of silica, 10% MeOH/DCM) to give 558mgs (74% yield) of 7.20 as a white foam. Usually the crude product wasof sufficient purity (by TLC and ¹H NMR) to use in the next reactionwithout purification by flash chromatography. [α]_(D) ²³+9.300 (c 0.57,CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ7.03 (d, 2H, J=8.1 Hz), 6.70 (d, 2H,J=8.1 Hz), 4.77 (bs, 1H), 4.18 (bs, 1H), 3.78 (bs, 1H), 3.50 (bs, 1H),2.82 (m, 2H), 2.71 (m, 2H), 2.45 (m, 2H), 1.79 (m, 1H), 1.39 (s, 9H),0.92 (d, 6H, J=6.6 Hz). ¹³C NMR: (CDCl₃) δ156.3, 154.9, 130.5, 115.5,70.4, 57.8, 35.6, 28.3, 28.0, 20.5. FABMS (C₁₉H₃₂N₂O₄) found 353.2.

(2S,3S)-N-Boc-3-amino-2-hydroxy-4-(4-t-butyldimethylsilyloxyphenyl)-1-N-(4carbobenzyloxyaminophenylsulfonyl)-(isobutyl)aminobutane7.23

A solution of hydroxyethylamine 7.20 (208 mg, 0.59 mmol) and TBS-Cl (179mg, 1.18 mmol) in THF (5.9 ml) was treated with imidazole (80 mg, 1.18mmol) and the mixture was stirred for 2 hours at room temperature. Afterdiluting with EthOAc (25 ml), the solution was washed with brine (2×),dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue wasdissolved in THF (5.9 ml) and NMM (0.200 ml, 1.83 mmol) and4-((carbobenzyloxy)-amino)-phenylsulfonyl chloride (211 mg, 0.648 mmol)was added to the reaction mixture. After stirring overnight at roomtemperature, the reaction was diluted with EthOAc (25 ml), washed withKHSO₄, NaHCO₃, H₂O and brine, and the phases were separated the organiclayer was dried over Na₂SO₄, filtered, and concentrated in vacuo, andthe residue was purified by flash chromatography (30% EthOAc/hexane) togive 258 mgs (59% yield) of 7.23 as a white foam. R_(f)0.47 (40%EtOAc/hexane). [α]_(D) ²³=+26.7 (c 0.58, CHCl₃). ¹H NMR (300 MHz, CDCl₃)δ7.53 (d, 2H, J=8.8 Hz), 7.34 (d, 2H, J=8.8 Hz), 7.26-7.16 (m, 5H), 6.91(d, 2H, J=8.4 Hz), 6.76 (s, 1H), 6.59 (d, 2H, J=8.4 Hz), 5.05 (s, 2H),4.47-4.35 (m, 1H), 3.72 (bs, 1H), 3.65-3.45 (m, 2H), 2.95-2.85 (m, 2H),2.82-2.58 (m, 4H), 1.65 (m, 1H), 1.18 (s, 9H), 0.80 (s, 9H), 0.71 (d,3H, J=6.61 Hz), 0.68 (d, 3H, J=6.6 Hz), 0.00 (s, 6H). ¹³C NMR:(CDCl₃)δ130.4, 128.8, 128.7, 128.6, 128.4, 120.1, 118.1, 67.5, 28.3, 27.1,25.7, 20.1, 19.9. FABMS (C₃₉H₅₇N₃O₈SiS) found 756 (M+H⁺).

(2S,3S)-N-Boc-3-amino-2-hydroxy-4-(4-ethylacetoxyphenyl)-1-N-(4-carbobenzyloxyaminophenylsulfonyl)-isobutylaminobutane7.24

A solution of 7.23 (248 mg, 0.327 mmol) in THF (5.5 ml) was treated withHF/pyridine (4.5 ml of a stock solution prepared from 1 g HF/pyridine, 2ml pyridine, and 8 ml THF). After stirring the reaction mixture at roomtemperature for 2.5 hours, at which time TLC indicated completedisappearance of starting material, the reaction was added dropwise to asolution of NaHCO₃ (50 ml). The mixture was extracted with DCM (3×25ml), and the combined organic phases were washed with KHSO₄ and brine,dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue wasdissolved in dioxane (3.3 ml) and Cs₃CO₃ (425 mg, 1.31 mmol) and ethylbromoacetate (0.218 ml, 1.96 mmol) was added to the reaction mixture.The reaction was stirred at 40-50° C. until TLC showed completedisappearance of starting material (ca. 3 hours), and then diluted withEthOAc. The mixture was washed with KHSO₄ and brine, dried over Na₂SO₄,filtered, and concentrated in vacuo. The residue was purified by flashchromatography (30-40-50% EthOAc/hexane) to give 170 mgs (71% yield) ofa foam. R_(f)0.25 (40% EtOAc/hexane). [α]_(D) ²³=+11.960 Cc 0.46,CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ7.64 (d, 2H, J=8.8 Hz), 7.54 (d, 2H,J=8.8 Hz), 7.43-7.34 (m, 51-I), 7.21 (s, 1H), 7.17 (d, 2H, J=8.6 Hz),6.84.(d, 2H, J=8.6 Hz), 5.28 (s, 2H), 4.67 (m, 1H), 4.59 (s, 2H), 4.25(q, 2H, J=7.1 Hz), 3.89-3.65 (m, 3H), 3.10-2.83 (m, 5H), 2.76 (dd, 1H,J=13.6, 7.0 Hz), 1.84 (m, 1H), 1.37 (s, 9H), 1.27 (t, 3H, J=7.13 Hz),0.90 (d, 3H, J=6.6 Hz), 0.85 (d, 3H J=6.6 Hz). FABMS C₃₇H₄₉N₃O₁₀S) found727 (M+H⁺).

N-{(S)-3-hydroxytetrahydrofuryloxycarbonyl}-(2S,3S)-3-amino-2-hydroxy-4-(4-ethylacetoxyphenyl)-1-N-(4-carbobenzyloxyaminophenylsulfonyl)-isobutylaminobutane7.25

The Boc group was cleaved from hydroxyethylamine 7.24 (150 mg, 0.206mmol) in HCl/dioxane (3 ml, 4 N) via general procedure D to give the HClsalt. A solution of the HCl salt in 50:50 dioxane/CH₃CN (3 ml) wastreated with TEA (0.068 ml, 0.618 mmol) and 3(S)-tetrahydrofurylp-nitrophenolcarbonate (57 mg, 0.226 mmol) and the reaction was stirredat room temperature for 1 hour and then at 50° C. for 2 hours. Afterdiluting with EthOAc (25 ml), the solution was washed with NaHCO₃ (10×),KHSO₄ (2×), an brine (2×), dried over Na₂SO₄, filtered, and concentratedin vacuo. The residue was purified by flash chromatography (30-40-50%EthOAc/hexane) to give 90 mgs (59% yield) of 7.25 as a white foam.R_(f)0.38 (70% EtOAc/hexane). [α]_(D) ²³=+2.710 (c 1.07, CHCl₃). ¹H NMR(300 MHz, CDCl₃) δ7.64 (d, 2H, J=8.8 Hz), 7.55 (d, 2H, J=9.0 Hz),7.44-7.34 (m, 5H), 7.28 (s, 1H), 7.14 (d, 2H, J=8.5 Hz), 6.83 (d, 2H,J=8.6 Hz), 5.22 (s, 2H), 5.14 (bs, 1H), 4.90 (m, 1H), 4.59 (s, 2H), 4.25(q, 2H, J=7.1 Hz), 3.90-3.73 (m, 6H), 3.69 (d, 1H, J=11.5 Hz), 3.13-2.82(m, 5H), 2.74 (dd, 1H, J=13.3, 6.4 Hz), 2.10 (m, 1H), 1.95 (m, 1H), 1.82(m, 1H), 1.28 (t, 3H, J=7.1 Hz), 0.90 (d, 3H, J=6.5 Hz), 0.86 (d, 3H,J=6.6 Hz). FABMS C₃₇H₄₇N₃O₁₁S) found 742 (M+H⁺).

Compound 2.6

A solution of 7.25 (19 mg, 0.0256 mmol) and Pd(OH)₂ (5 mg) in MeOH (1ml) was evacuated three times and each time the vacuum was broken withhydrogen (1 atm). After the third time, the reaction was stirred underhydrogen for 1 hour, filtered over celite, and concentrated in vacuo togive 16 mgs (100% yield) of a white film which was pure by TLC.R_(f)=0.4 (10:90) MeOH/DCM. ¹H NMR (300 MHz, CDCl₃) δ7.42 (d, 2H, J=8.05Hz), 7.08 (d, 2H, J=8.05 Hz), 6.77 (d, 2H, J=8.04 Hz), 6.60 (d, 2H,J=8.2 Hz), 5.06 (bs, 1H), 4.83 (m, 1H), 4.53 (s, 2H), 4.20 (q, 2H, J=7.1Hz), 3.82-3.55 (m, 71-1), 3.00 (m, 1H), 2.92-2.60 (m, SH), 2.02 (m, 1H),1.87 (m, 1H), 1.73 (m, 1H), 1.24 (t3H, J=7.1 Hz), 0.84 (d, 3H, J=6.28Hz), 0.79 (d, 3H, J=6.43 Hz).

Compound 2.6-(MeLeu(3-OH)¹, (D)MeSer³, Lys⁷)-CsA conjugate 2.10

A solution of ester 7.25 (36 mg, 0.0485 mmol) in MeOH/THF (1:1 0.480 ml)was treated with LiOH (0.1 ml, 1 N LiOH (aq)) and the solution wasstirred for 3 hours, at which time an additional equivalent of LiOH wasadded and the reaction was stirred for another hour. The reaction wasconcentrated in vacuo, dissolved in H₂O (20 ml) which was washed withether (15 ml), acidified with 1 N KHSO₄, and extracted with DCM (2×) andEthOAc (3×). The combined organic layers were dried over Na₂SO₄,filtered, concentrated in vacuo to give acid 7.26 which was useddirectly in the next coupling reaction.

The Cbz-protected carboxylic acid derivative 7.26 (25 mg, 0.035 mmol)and the free amine CsA analog 4.11 (44 mg, 0.035 mmol, produced from4.20 via general procedure K) were dissolved in DCM (1 ml). PyAOP (20mg, 0.0385 mmol) and DIEA (0.0128 ml, 0.0735 mmol) were addedsequentially to the solution and the reaction was stirred overnight atroom temperature. After diluting with EthOAc, the mixture was washedwith H₂O (2×), dried over Na₂SO₄, and concentrated in vacuo. The residuewas purified by flash chromatography (2-3-4-5% MeOH/DCM) to give 34 mg(50% yield) of conjugate 8.2. R_(f)=0.62 (9:1 DCM:MeOH). Conjugate 8.2(25 mg, 0.0128 mmol) was dissolved in MeOH (1 ml) and Pd(OH)₂ (5 mg) wasadded to the solution. The reaction vessel was evacuated and the vacuumwas broken each time with hydrogen (1 atm). After repeating thisevacuation sequence three times, the reaction was stirred overnightunder an atmosphere of hydrogen, filtered through an acrodisk, andconcentrated in vacuo to give 22 mg (100% yield) of fully deprotectedconjugate 2.10 which was pure by TLC. R_(f)=0.59 (9:1 DCM/MeOH). FABMSC₉₀H₁₅₁N₁₅O₂₁S) found 1811.2.

Examples 6-10 Anti-HIV Activity of the CsA Conjugates

The conjugates 2.9 and 2.10, and the Cbz-protected CsA analog(MeLeu(3-OH)¹, (D)MeSer³, Lys(2Cl-Cbz)⁷)-CsA 4.20 were sent to theNational Cancer Institute for testing according to the NCI's “In VitroAnti-AIDS Drug Discovery Program.” The compounds 2.6 and 1.13 (an analogof VX-478) were also sent for comparative evaluation. The procedure usedin the National Cancer Institute's test for agents active against humanimmunodeficiency virus (HIV) is designed to detect agents acting at anystage of the virus reproductive cycle. The assay basically involves thekilling of T4 lymphocytes by HIV. Small amounts of HIV are added tocells, and two cycles of virus reproduction are necessary to obtain therequired cell killing. Agents that interact with virions, cells, orvirus gene-products to interfere with viral activities will protectcells from cytolysis. The system is automated in several features toaccommodate large numbers of candidate agents and is generally designedto detect anti-HIV activity. However, compounds that degenerate or arerapidly metabolized in the culture conditions may not show activity inthis screen. All tests are compared with at least one positive (e.g.,AZT-treated) control done at the same time under identical conditions.

The Procedure:

1. Candidate agent is dissolved in dimethyl sulfoxide (unless otherwiseinstructed) then diluted 1:100 in cell culture medium before preparingserial half-log₁₀ dilutions. T4 lymphocytes (CEM cell line) are addedand after a brief interval HIV-1 is added, resulting in a 1:200 finaldilution of the compound. Uninfected cells with the compound serve as atoxicity control, and infected and uninfected cells without the compoundserve as basic controls.

2. Cultures are incubated at 37° in a 5% carbon dioxide atmosphere for 6days.

3. The tretrazolium salt, XTT, is added to all wells, and cultures areincubated to allow formazan color development by viable cells.

4. Individual wells are analyzed spectrophotometrically to quantitateformazan production, and in addition are viewed microscopically fordetection of viable cells and confirmation of protective activity.

5. Drug-treated virus-infected cells are compared with drug-treatednoninfected cells and with other appropriate controls (untreatedinfected and untreated noninfected cells, drug-containing wells withoutcells, etc.) on the same plate.

6. Data are reviewed in comparison with other tests done at the sametime and a determination about activity is made.

(See Weislow, O. W., Kiser, R., Fine, D., Bader, J., Showmaker, R. H.,Boyd, M. R.: New soluble-formazan assay for HIV-1 cytopathic effects:application to high-flux screening of synthetic and natural products forAIDS-antiviral activity. J. Natl. Cancer Inst. (1989) 81:577-586)

Examples 6, 7, and 8

Compounds 2.8, 2.10, and 4.20 were submitted to the NCI for HIV testingusing the above protocol. Each sample was tested in quadruplicate runsof the assay. Representative graphs of the results are presented inFIGS. 1, 3, and 4, respectively. In each of the graphs, the solid linerepresents the growth of an HIV-infected culture which has been treatedwith one of the test compounds. The dashed line represents the growth ofcontrol culture. The control comprises an uninfected culture which hasalso been treated with the test compound.

Conjugate 2.8, the results for which are depicted in FIG. 1, was“confirmed active” against HIV. Conjugate 2.10, the results for whichare depicted in FIG. 3, was also “confirmed active” against HIV. CsAanalog 4.20, the results for which are depicted in FIG. 4, was“confirmed moderate” against HIV.

These results clearly show that the subject compounds are active in theinhibition of HIV replication and thus are useful in the prevention andtreatment of HIV-mediated disorders.

Example 9

Compound 2.6 was subjected to the NCI anti-HIV assay described above.The results are presented in FIG. 2. Compound 2.6 was “confirmedmoderate” in its activity against HIV.

These Examples show that the present invention provides a generalapproach for conjugating anti-HIV compounds to CsA in order to increasetheir bioavailability without having an adverse impact on their abilityto inhibit HIV replication.

The invention is not confined to the particular reagents, reactions,compounds, and conjugates disclosed above, but embraces all forms andequivalents thereof as come within the scope of the following claims.

26 1 11 PRT Artificial Sequence MOD_RES (1) MeBmt = (4R)-4-[(E)-2-butenyl]-4-N-methyl-(L)-threonine 1 Xaa Xaa Xaa Xaa Val XaaAla Ala Xaa Xaa Xaa 1 5 10 2 4 PRT Artificial Sequence MOD_RES (1)MeLeu; BLOCKED 2 Xaa Val Xaa Lys 1 3 5 PRT Artificial Sequence MOD_RES(1) MeSer(Obn) = benzyloxy-protected N-methyl- serine; BLOCKED 3 Xaa XaaVal Xaa Lys 1 5 4 6 PRT Artificial Sequence MOD_RES (1) BLOCKED, Abu 4Xaa Xaa Xaa Val Xaa Lys 1 5 5 7 PRT Artificial Sequence MOD_RES (1)MeLeu(3-OH) = 3-hydroxy-N-methyl-leucine; BLOCKED 5 Xaa Xaa Xaa Xaa ValXaa Lys 1 5 6 7 PRT Artificial Sequence MOD_RES (1) MeLeu(3-OH) =3-hydroxy-N-methyl-leucine 6 Xaa Xaa Xaa Xaa Val Xaa Lys 1 5 7 11 PRTArtificial Sequence MOD_RES (1) (D) Ala; BLOCKED 7 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Val Xaa Lys 1 5 10 8 4 PRT Artificial Sequence MOD_RES (1)MeAla = N-methyl-alanine; BLOCKED 8 Xaa Val Xaa Lys 1 9 11 PRTArtificial Sequence MOD_RES (1) MeuLeu(3-OH) =3-hydroxy-N-methyl-leucine 9 Xaa Xaa Xaa Xaa Val Xaa Lys Ala Xaa Xaa Xaa1 5 10 10 5 PRT Artificial Sequence MOD_RES (1) (D)MeSer(OTBS) =(D)-N-methyl-serine, tert-butyldimethylsilyloxy-protected; BLOCKED 10Xaa Xaa Val Xaa Lys 1 5 11 5 PRT Artificial Sequence MOD_RES (1)(D)MeSer = (D)-N-methyl-serine; BLOCKED 11 Xaa Xaa Val Xaa Lys 1 5 12 6PRT Artificial Sequence MOD_RES (1) Abu; BLOCKED 12 Xaa Xaa Xaa Val XaaLys 1 5 13 7 PRT Artificial Sequence MOD_RES (1) MeLeu(3-OH) =3-hydroxy-N-methyl-leucine; BLOCKED 13 Xaa Xaa Xaa Xaa Val Xaa Lys 1 514 7 PRT Artificial Sequence MOD_RES (1) MeLeu(3-OH) =3-hydroxy-N-methyl-leucine 14 Xaa Xaa Xaa Xaa Val Xaa Lys 1 5 15 11 PRTArtificial Sequence MOD_RES (1) (D)-Ala; BLOCKED 15 Ala Xaa Xaa Xaa XaaXaa Xaa Xaa Val Xaa Lys 1 5 10 16 11 PRT Artificial Sequence MOD_RES (1)MeLeu(3-OH) = 3-hydroxy-N-methyl-leucine 16 Xaa Xaa Xaa Xaa Val Xaa LysAla Xaa Xaa Xaa 1 5 10 17 6 PRT Artificial Sequence MOD_RES (1) BLOCKED,Abu 17 Xaa Xaa Xaa Val Xaa Lys 1 5 18 7 PRT Artificial Sequence MOD_RES(1) MeLeu(3-OH) = 3-hydroxy-N-methyl-leucine; BLOCKED 18 Xaa Xaa Xaa XaaVal Xaa Lys 1 5 19 7 PRT Artificial Sequence MOD_RES (1) MeLeu(3-OH) =3-hydroxy-N-methyl-leucine 19 Xaa Xaa Xaa Xaa Val Xaa Lys 1 5 20 11 PRTArtificial Sequence Description of Artificial Sequence synthesizedpolypeptide 20 Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Lys 1 5 10 21 11PRT Artificial Sequence Description of Artificial Sequence synthesizedpolypeptide 21 Xaa Xaa Xaa Xaa Val Xaa Lys Ala Xaa Xaa Xaa 1 5 10 22 5PRT Artificial Sequence Description of Artificial Sequence synthesizedpolypeptide 22 Phe Xaa Pro Ile Phe 1 5 23 6 PRT Artificial SequenceDescription of Artificial Sequence synthesized polypeptide 23 Asn PheXaa Pro Ile Phe 1 5 24 6 PRT Artificial Sequence Description ofArtificial Sequence synthesized polypeptide 24 Xaa Xaa Xaa Val Xaa Ala 15 25 7 PRT Artificial Sequence Description of Artificial Sequencesynthesized polypeptide 25 Xaa Xaa Xaa Xaa Val Xaa Ala 1 5 26 4 PRTArtificial Sequence Description of Artificial Sequence synthesizedpolypeptide 26 Ala Xaa Xaa Xaa

What is claimed is:
 1. Non-immunosupressive cyclosporins comprising acyclic undecapeptide of Formula I:

wherein V is a MeLeu(3-OH), MeLeu, MeSer, MeSer-PG, MeThr, or MeThr-PGresidue; W is a (D)-N-methyl-amino acid or an N-methylglycyl residue; Xand X′ are independently an N-methyl-leucinyl or an N-methylalanylresidue; Y is a lysyl, homo-lysyl, ornithinyl, lysyl-PG, homo-lysyl-PG,or ornithinyl-PG residue; wherein each PG is, independently, aside-chain protecting group; and Z is absent or is an HIV proteaseinhibitor moiety conjugated to Y via a side-chain on Y and selected fromthe group consisting of

wherein R is Ac-Ser-Leu-Asn; and salts thereof.
 2. The cyclosporins ofclaim 1, wherein Z is absent.
 3. The cyclosporins of claim 1, whereineach PG is independently selected from the group consisting of allyl,benzyl, benzyloxy, 2-chloro-benzyloxy, and combinations thereof.
 4. Thecyclosporins of claim 1, wherein Y is a lysyl residue.
 5. Thecyclosporins of claim 4, wherein V is a 3-hydroxy-N-methyl-leucineresidue, W is a N-methylglycyl, (D)-N-methylserinyl, or(D)-N-methylserinyl-PG residue, wherein PG is selected from the groupconsisting of allyl, benzyl, benzyloxy, and 2-chloro-benzyloxy.
 6. Thecyclosporins of claim 4, wherein X and X′ are N-methylalanyl residues.7. The cyclosporins of claim 4, wherein X and X′ are N-methyl-leucineresidues.
 8. The cyclosporins of claim 4, wherein Z is selected from thegroup consisting of

wherein R is Ac-Ser-Leu-Asn.
 9. The cyclosporins of claim 8 wherein W isa (D)-N-methylserinyl or a (D)-N-methylserinyl-PG residue, wherein PG isselected from the group consisting of allyl, benzyl, benzyloxy, and2-chloro-benzyloxy; X and X′ are N-methyl-leucine residues; and Z is

wherein R is Ac-Ser-Leu-Asn.
 10. The cyclosporins of claim 8 wherein Wis a (D)-N-methylserinyl or a (D)-N-methylserinyl-PG residue, wherein PGis selected from the group consisting of allyl, benzyl, benzyloxy, and2-chloro-benzyloxy; X and X′ are N-methyl-leucine residues; and Z is


11. The cyclosporins of claim 8 wherein W is a (D)-N-methylserinyl or a(D)-N-methylserinyl-PG residue, wherein PG is selected from the groupconsisting of allyl, benzyl, benzyloxy, and 2-chloro-benzyloxy; X and X′are N-methyl-leucine residues; and Z is


12. The cyclosporins of claim 8 wherein W is a (D)-N-methylserinyl or a(D)-N-methylserinyl-PG residue, wherein PG is selected from the groupconsisting of allyl, benzyl, benzyloxy, and 2-chloro-benzyloxy; X and X′are N-methyl-leucine residues; and Z is


13. The cyclosporins of claim 8 wherein W is a (D)-N-methylserinyl or a(D)-N-methylserinyl-PG residue, wherein PG is selected from the groupconsisting of allyl, benzyl, benzyloxy, and 2-chloro-benzyloxy; and Xand X′ are N-methylalanyl residues.
 14. A compound comprising aconjugate produced by: (a) forming a cyclosporin analog of formula

wherein V is a MeLeu(3-OH), MeLeu, MeSer, MeSer-PG, MeThr, or MeThr-PGresidue, wherein each PG is, independently, a side-chain protectinggroup; W is a (D)-N-methyl-amino acid residue or an N-methylglycylresidue; X and X′ are independently an N-methyl-leucinyl or anN-methylalanyl residue; and Y is a lysyl, is formed; and comprising alysyl residue having an ε-aminobutyl side-chain at position-7 of thecyclosporin analog; and (b) conjugating an HIV protease inhibitor, Z, tothe ε-aminobutyl side-chain of the lysyl residue, Z being selected fromthe group consisting of

wherein R is Ac-Ser-Leu-Asn; and wherein the compound simultaneouslybinds to and inhibits the action of cyclophilin and HIV protease. 15.The compound of claim 14, wherein in step (a) a cyclosporin analogwherein V is a MeLeu(3-OH) residue; W is a N-methylglycyl or(D)-N-methylserinyl residue; and X and X′ are N-methyl-leucine residuesis formed; and in step (b), Z, an HIV protease inhibitor moiety offormula

wherein R is Ac-Ser-Leu-Asn, is conjugated to Y.
 16. The compound ofclaim 14, wherein in step (a) a cyclosporin analog wherein V is aMeLeu(3-OH) residue; W is a N-methylglycyl or (D)-N-methylserinylresidue; and X and X′ are N-methyl-leucine residues is formed; and instep (b), Z, an HIV protease inhibitor moiety of formula

 is conjugated to Y.
 17. The compound of claim 14, wherein in step (a) acyclosporin analog wherein V is a MeLeu(3-OH) residue; W is aN-methylglycyl or (D)-N-methylserinyl residue; and X and X′ areN-methyl-leucine residues is formed; and in step (b), Z, an HIV proteaseinhibitor moiety of formula

 is conjugated to Y.
 18. The compound of claim 14, wherein in step (a) acyclosporin analog wherein V is a MeLeu(3-OH) residue; W is aN-methylglycyl or (D)-N-methylserinyl residue; and X and X′ areN-methyl-leucine residues is formed; and in step (b), Z, an HIV proteaseinhibitor moiety of formula

 is conjugated to Y.
 19. The compound of claim 14, wherein in step (a) acyclosporin analog wherein X and X′ are N-methylalanyl residues isformed.
 20. A pharmaceutical composition for the treatment ofHIV-mediated disorders in humans comprising an effective HIVprotease-inhibiting amount of a compound of claim 1 or apharmaceutically-acceptable salt thereof in combination with apharmaceutically-acceptable liquid or solid carrier.
 21. Thepharmaceutical composition of claim 20 comprising a compound wherein Yis a lysyl residue; and Z is selected from the group consisting of

wherein R is Ac-Ser-Leu-Asn.
 22. The composition of claim 21, wherein Wis a (D)-N-methylserinyl or a (D)-N-methylserinyl-PG residue, wherein PGis selected from the group consisting of allyl, benzyl, benzyloxy, and2-chloro-benzyloxy; and X and X′ are N-methyl-leucinyl residues.
 23. Thecomposition of claim 21, wherein wherein W is a (D)-N-methylserinyl or a(D)-N-metbylserinyl-PG residue, wherein PG is selected from the groupconsisting of allyl, benzyl, benzyloxy, and 2-chloro-benzyloxy; and Xand X′ are N-methylalanyl residues.
 24. A pharmaceutical composition forthe prevention and treatment of HIV-mediated disorders in humanscomprising an effective HIV protease-inhibiting amount of a compound ofclaim 14 or a pharmaceutically-acceptable salt thereof in combinationwith a pharmaceutically-acceptable liquid or solid carrier.
 25. A methodof treating HIV-mediated disorders in humans comprising administering toa human subject in need thereof an effective HIV protease-inhibitingamount of one or more compounds of claim 1, orpharmaceutically-acceptable salts thereof.
 26. The method of claim 25,wherein one or more compounds selected from the group consisting of:

wherein V is a MeLeu(3-OH) residue; wherein W is an N-methylglycyl,(D)-N-methylserinyl, or (D)-N-methylserinyl-PG residue, wherein each PGis, independently, a side-chain protecting group; X and X′ areindependently an N-methyl-leucinyl or an N-methylalanyl residue; Y is alysyl residue; and Z is selected from the group consisting of

wherein R is Ac-Ser-Leu-Asn; and pharmaceutically-acceptable saltsthereof, is administered.
 27. The method of claim 26, wherein a compoundwhere Z is

wherein R is Ac-Ser-Leu-Asn, is administered.
 28. The method of claim26, wherein a compound where Z is

is administered.
 29. The method of claim 24, wherein a compound where Zis

is administered.
 30. The method of claim 24, wherein a compound where Zis

is administered.
 31. A method of preventing and treating HIV-mediateddisorders in humans comprising administering to a human subject in needthereof an effective HIV protease-inhibiting amount of one or morecompounds of claim 13, or pharmaceutically-acceptable salts thereof.